Method for treating cellulosic material and CBHII/CEL6A enzymes useful therein

ABSTRACT

The present invention relates to the production of sugar hydrolysates from cellulosic material. The method may be used, for example for producing fermentable sugars for the production of bioethanol from lignocellulosic material. Cellulolytic enzymes and their production by recombinant technology are described, as well as uses of the enzymes and enzyme preparations.

FIELD OF THE INVENTION

The present invention relates to a method for treating cellulosicmaterial with a fungal CBHII/Cel6A cellobiohydrolase enzyme or an enzymepreparation comprising said enzyme. The enzyme is useful in variousindustrial applications, particularly in production of biofuels, whereproduction of fermentable sugars from lignocellulosic material atmoderate to elevated temperature ranges is advantageous. The inventionfurther relates to fungal CBHII/Cel6A polypeptides and isolated nucleicacid molecules encoding said enzymes, a recombinant vector, host cellsfor producing said enzymes, enzyme compositions comprising said enzymesas well as a process for preparing such compositions. The inventionrelates also to various uses of said enzymes or enzyme compositions, inwhich enzymatic conversion of cellulosic or lignocellulosic material isdesired.

BACKGROUND OF THE INVENTION

Limited resources of fossil fuels and increasing amounts of CO₂ releasedfrom them and causing the greenhouse phenomenon have raised a need forusing biomass as a renewable and clean source of energy. One promising,alternative technology is the production of biofuels, such as ethanol,butanol or propanol from cellulosic materials. In the transportationsector biofuels are for the time being the only option, which couldreduce the CO₂ emissions by an order of magnitude. The ethanol can beused in existing vehicles and distribution systems and thus it does notrequire expensive infrastructure investments. Sugars derived fromcellulosic and lignocellulosic renewable raw materials can also be usedas raw materials for a variety of chemical products that can replaceoil-based chemicals.

Most of the carbohydrates in plants are in the form of lignocellulose,which essentially consists of cellulose, hemicellulose and lignin. In aconventional lignocellulose-to-ethanol process the lignocellulosicmaterial is first pretreated either chemically or physically, using acidhydrolysis, steam explosion, ammonia fiber expansion, alkaline wetoxidation or ozone pretreatment, to make the cellulose fraction moreaccessible to enzymatic hydrolysis. The cellulose fraction is thenhydrolyzed to obtain sugars that can be fermented by yeast into ethanoland distilled to obtain pure ethanol. Lignin is obtained as a mainco-product that may be used as a solid fuel. In this separate hydrolysisand fermentation (SHF) process the temperature of enzymatic hydrolysisis typically higher than that of fermentation. The use of thermostableenzymes in hydrolysis offer potential benefits, such as higher reactionrates at elevated temperatures, reduction of enzyme load due to higherspecific activity and life-time of enzymes, increased flexibility withrespect to process configuration and better hygiene.

There is continuous research for making the bioethanol productionprocess more economical. One of the options is the simultaneoussaccharification and fermentation (SSF) process. The principal benefitsare the reduced end-product inhibition of the enzymatic hydrolysis andthe reduced investment costs. The challenges are in finding favorableconditions, e.g. temperature and pH, for both the enzymatic hydrolysisand fermentation. In the consolidated bioprocess (CBP), the amount ofexternally added enzymes can be significantly reduced by exploiting afermentative organism or ethanolgen, which is capable of producing a setof lignocellulolytic enzymes.

In recent years, metabolic engineering for microorganisms used inethanol production has shown significant progress. Besides Saccharomycescerevisiae, microorganisms such as the bacterial species Zymomonas andEscherichia coli and yeasts such as Pichia stipitis and Kluyveromycesfragilis have been targeted for ethanol production from cellulose. Inthe SSF process, the inhibitor and temperature tolerance as well as theability to utilize multiple sugars are important properties of thefermenting microorganism. Engineered yeasts have been developed that areable to ferment pentose sugars xylose and arabinose in addition toglucose. Thermophilic microbes, like Thermoanaerobacteriumsaccharolyticum or Clostridium thermocellum have been engineered toferment sugars, including xylose, to ethanol at elevated temperatures of50° C.-60° C. (thermophilic SSF or TSSF). Such fermentative organismshave also potential as CBPs (Shaw et al. 2008).

Enzymatic hydrolysis is considered the most promising technology forconverting cellulosic biomass into fermentable sugars. However,enzymatic hydrolysis is used only to a limited amount at industrialscale, and especially when using strongly lignified material such aswood or agricultural waste the technology is not satisfactory. Effortshave been made to improve the efficiency of the enzymatic hydrolysis ofthe cellulosic material (Badger 2002; Kurabi et al., 2005).

WO2001060752 (Forskningscenter Risø, DK) describes a continuous processfor converting solid lignocellulosic biomass into combustible fuelproducts. After pretreatment by wet oxidation or steam explosion thebiomass is partially separated into cellulose, hemicellulose and lignin,and is then subjected to partial hydrolysis using one or morecarbohydrase enzymes (EC 3.2).

WO2002024882 (Iogen Bio-Products Corp., CA) pertains a method ofconverting cellulose to glucose by treating a pretreated lignocellulosicsubstrate with an enzyme mixture comprising cellulase and a modifiedcellobiohydrolase I (CBHI) obtained by inactivating its cellulosebinding domain (CBD). US2004/0005674 (Athenix Corp., US) describes novelenzyme mixtures that can be used directly on lignocellulose substrate.The synergistic enzyme mixture contains a cellulase and an auxiliaryenzyme such as cellulase, xylanase, ligninase, amylase, protease,lipidase or glucuronidase, or any combination thereof. Cellulase isconsidered to include endoglucanase (EG), beta-glucosidase (BG) andcellobiohydrolase (CBH) enzymes.

US20050164355 (Novozymes Biotech Inc., US) describes a method fordegrading lignocellulosic material with one or more cellulolytic enzymesselected from EG, BG and CBH and in the presence of at least onesurfactant. Additional enzymes such as hemicellulases, esterase,peroxidase, protease, laccase or mixture thereof may also be used.

The best-investigated and most widely applied cellulolytic enzymes offungal origin have been derived from Trichoderma reesei (the anamorph ofHypocrea jecorina). Cellulases from less known fungi have also beendisclosed.

Hong et al. (2003a and 2003b) characterize EG and CBHI of Thermoascusaurantiacus and their production in yeast. Tuohy et al. (2002) describethree forms of cellobiohydrolases, including CBHI and CBHII fromTalaromyces emersonii.

Use of cellobiohydrolase I (CBHI), a member of family 7 of glycosylhydrolases in enzymatic conversion of cellulosic material is known, forexample from WO03/000941 (Novozymes A/S, DK), which relates to CBHIenzymes obtained from various fungi. WO2005074656 (Novozymes Inc., US)discloses polypeptides having cellulolytic activity derived e.g. fromThermoascus aurantiacus.

WO2007071818 (Roal Oy, FI) describes production of sugar hydrolysatesfrom cellulosic material by enzymatic conversion and enzyme preparationscomprising said enzymes. Enzymes useful in the method includethermostable cellobiohydrolase, endoglucanase, beta-glucosidase andoptionally xylanase deriving from Thermoascus aurantiacus, Acremoniumthermophilum or Chaetomium thermophilum.

Cellobiohydrolases II have been disclosed in several applications.WO2004056981 (Novozymes A/S, DK) discloses polypeptides havingcellobiohydrolase II activity and polynucleotides encoding thepolypeptides as well as methods for producing and using the polypeptidesin applications, such as in production of ethanol. Full length DNAsequences are disclosed from Aspergillus tubigensis, Chaetomiumthermophilum, Myceliophtora thermophila, species of Thielavia,Acremonium thermophilum, Trichophaea saccata, Stibella anualata andMalbrancheae cinnamonea. EP1578964 B1 (Novozymes A/S, DK) discloses thefull length amino acid sequence of C. thermophilum CBHII and apolypeptide encoded by a nucleotide sequence hybridizing under stringentconditions with a fragment of the nucleotide sequence encoding saidenzyme.

CN1757709 (Shandong Agricultural Univ., CN) discloses the nucleotidesequence of a thermophilic CBHII enzyme of Chaetomium thermophilum CT2and its expression in Pichia pastoris yeast. The enzyme is capable ofconverting the rejected fiber material.

WO2006074005 (Genencor Int., Inc., US) discloses a variant of Hypocreajecorina (Trichoderma reesei) CBHII/Cel6A enzyme. The variant enzyme isuseful, for example in bioethanol production.

WO2007094852 (Diversa Corp., US; Verenium Corp., US) disclosescellulolytic enzymes, nucleic acids encoding them and methods for theirproduction and use. The enzyme may be an endoglucanase, acellobiohydrolase, a beta-glucosidase, a xylanase, a mannanase, abeta-xylosidase, an arabinofuranosidase or an oligomerase. The enzymeand enzyme mixtures are useful, for example in making fuel orbioethanol.

WO2008095033 (Syngenta, CH, Verenium Corp., US) discloses enzymes havinglignocellulolytic activity, including cellobiohydrolases useful, forexample in making fuels and processing biomass materials. WO2009045627(Verenium Corp., US) discloses methods for breaking down hemicelluloseby using enzymes having xylanase, mannanase and/or glucanase activityand increased activity and stability at increased pH and temperature.

WO2009089630 (Iogen Energy Corp., CA) discloses a variant of a family 6cellulase with reduced inhibition by glucose, comprising one or moreamino acid substitutions.

WO2009059234 (Novozymes Inc., US) discloses methods of producingcellulosic material reduced in a redox active metal ion useful indegrading or converting a cellulosic material and producing afermentation product. The application discloses, e.g. a CBHIIpolypeptide of Chaetomium thermophilum.

WO2009085868 (Novozymes A/S, DK) discloses polypeptides, polynucleotidesencoding the enzyme and a method for producing a fermentation product,comprising saccharification of a cellulosic material with a cellulolyticenzyme composition comprising said polypeptide. Such cellobiohydrolasesmay derive from Trichoderma reesei, Humicola insolens, Myceliophtorathermophila, Thielavia terrestris and Chaetomium thermophilum.

WO2006074435 and US2006218671 (Novozymes Inc., US) disclose nucleotideand amino acid sequences of Thielavia terrestris Cel6Acellobiohydrolase. U.S. Pat. No. 7,220,565 (Novozymes Inc., US)discloses polypeptides having cellulolytic enhancing activity andidentity to the mature amino acid sequence of Myceliophtora thermophilaCBHII.

US20070238155 and US20090280105 (Dyadic Int., Inc., US) disclose enzymecompositions comprising novel enzymes from Chrysosporium lucknowense,comprising e.g. the CBHIIa and CBHIIb enzymes assigned to family 6 ofglycosyl hydrolases. The enzyme compositions are effective in hydrolysisof the lignocellulosic material.

The genome sequence of Neurospora crassa OR74A is disclosed in Galaganet al. (2003), including a sequence of exoglucanase 2 precursor. Collinset al. (2003) disclose the coding sequence of Talaromyces emersoniiCBHII/Cel6A.

The market in biofuels such as renewable transportation fuels isexpected to increase considerably in near future. As a result, there isa rapidly growing interest in the use of alternative feedstock forbiofuel production. Fermentation of cellulosic biomass present in plantsand woods or municipal waste to ethanol and other alcohols is anattractive route to fuels that supplement fossil fuels. One barrier ofproduction of biofuels from cellulosic and lignocellulosic biomass isthe robustness of the cell walls and the presence of sugar monomers inthe form of inaccessible polymers that require a great amount ofprocessing to make the sugar monomers available to the microorganismsthat are typically used to produce alcohol by fermentation. Thus, thereis a continuous need for new methods as well as new enzymes and enzymemixtures, which enhance the efficiency of the degradation of thecellulosic and lignocellulosic substrates. Particularly, enzymes andenzyme mixtures are needed which are able to attack different glycosidiclinkages of the crystalline cellulosic material and thus provide almostcomplete hydrolysis of the varying materials to be treated. There isalso a need for enzymes, which are stable at elevated processtemperatures, thus enabling the use of high biomass consistency andleading to high sugar and ethanol concentrations. This approach may leadto significant savings in energy and investments costs. The hightemperature also decreases the risk of contamination during hydrolysis.The present invention aims to meet at least part of these needs.

SUMMARY OF THE INVENTION

The aim of the present invention is to provide new enzymes and enzymecompositions for enhancing the efficiency of cellulose hydrolysis. Thecellobiohydrolases and particularly cellobiohydrolases II obtainablefrom Acremonium thermophilum, Melanocarpus albomyces, Chaetomiumthermophilum or Talaromyces emersonii are useful in hydrolyzing anddegrading cellulosic material. The enzymes are kinetically veryeffective over a broad range of temperatures, and although they havehigh activity at high temperatures, they are also very efficient atstandard hydrolysis temperatures. This makes them extremely well suitedfor varying cellulosic substrate hydrolysis processes carried out bothat conventional temperatures and at elevated temperatures.

The present invention relates to a method for treating cellulosicmaterial with a CBHII/Cel6A polypeptide or an enzyme preparationcomprising said polypeptide or a fermentative microorganism producingsaid polypeptide, said method comprising the steps: i) production of aCBHII/Cel6A polypeptide of the invention or an enzyme preparationcomprising said polypeptide or a fermentative microorganism producingsaid polypeptide; ii) reacting the cellulosic material with theCBHII/Cel6A polypeptide of the invention or the enzyme preparationcomprising said polypeptide or the fermentative microorganism producingsaid polypeptide; and iii) obtaining partially or fully hydrolyzedcellulosic material. The CBHII/Cel6A polypeptide useful in said methodhas cellobiohydrolase activity and comprises an amino acid sequencehaving at least 76% identity to the full-length polypeptide of SEQ IDNO: 12, at least 76% identity to the full-length polypeptide of SEQ IDNO:14, at least 95% identity to the full-length polypeptide of SEQ IDNO:15, or at least 91% identity to the full-length polypeptide of SEQ IDNO:16. The CBHII/Cel6A polypeptide may also be a fragment or varianthaving similar properties, such as similar substrate specificity and pHand temperature dependence or stability. The CBHII/Cel6Acellobiohydrolase useful in the method is a cellobiohydrolase II enzymeof family 6 of glycosyl hydrolases and has both a conserved fold andstereochemistry of the hydrolysis reaction.

The CBHII/Cel6A cellobiohydrolases applicable in the method may beobtained from a genus of Acremonium, Melanocarpus, Chaetomium orTalaromyces, more preferably from A. thermophilum, M. albomyces, C.thermophilum or T. emersonii, most preferably from the deposited strainA. thermophilum CBS 116240, M. albomyces CBS 685.95, C. thermophilum CBS730.95 or T. emersonii DSM 2432.

The CBHII/Cel6A cellobiohydrolase applicable in the method is capable ofhydrolyzing different cellulosic materials at moderate to elevatedtemperatures, particularly in combination with other enzymes used inhydrolysis of various cellulosic or lignocellulosic materials.

The CBHII/Cel6A cellobiohydrolase of the invention is applicable invarious uses, particularly in production of biofuel,

The present invention relates also to novel CBHII/Cel6Acellobiohydrolases, which have cellobiohydrolase activity and comprisean amino acid sequence having at least 76% identity to the full-lengthpolypeptide of SEQ ID NO: 12, at least 76% identity to the full-lengthpolypeptide of SEQ ID NO: 14, at least 95% identity to the full-lengthpolypeptide of SEQ ID NO:15 or at least 91% identity to the full-lengthpolypeptide of SEQ ID NO:16. Said CBHII/Cel6A polypeptide may also be afragment or variant having similar properties, such as similar substratespecificity and pH and temperature dependence or stability. SaidCBHII/Cel6A cellobiohydrolase is capable of hydrolyzing cellulosicmaterial at moderate to elevated temperatures.

Said enzyme is encoded by an isolated nucleic acid molecule, whichcomprises a polynucleotide sequence encoding a polypeptide of theinvention. Preferably, said nucleic acid molecule comprises thepolynucleotide sequence defined in SEQ ID NO: 11, SEQ ID NO:13, SEQ IDNO:9 or SEQ ID NO:10 or a subsequence thereof.

The CBHII/Cel6A cellobiohydrolase of the invention may be encoded by anisolated nucleic acid molecule, which hybridizes under stringentconditions to a polynucleotide sequence included in SEQ ID NO:11, SEQ IDNO:13, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:7, SEQ ID NO:8 or asubsequence thereof.

Said enzyme is encoded by an isolated polynucleotide included in plasmidpALK2582 deposited in Escherichia coli under accession number DSM 22946,plasmid pALK2581 deposited in E. coli under accession number DSM 22945,plasmid pALK2904 deposited in E. coli under accession number DSM 22947or plasmid pALK3006 deposited in E. coli under accession number DSM23185

The CBHII/Cel6A cellobiohydrolase of the invention may be produced froma recombinant expression vector comprising the nucleic acid molecule ornucleotide sequence encoding said cellobiohydrolase. Saidcellobiohydrolase polypeptide may be produced in a heterologous host,preferably in a microbial host.

The invention relates also to an isolated nucleic acid moleculecomprising a polynucleotide sequence encoding a fungal CBHII/Cel6Apolypeptide selected from the group consisting of:

-   -   (a) a nucleic acid molecule or polynucleotide sequence encoding        a polypeptide having cellobiohydrolase activity and comprising        the full-length amino acid sequence as depicted in SEQ ID NO:12,        SEQ ID NO:14, SEQ ID NO:15 or SEQ ID NO:16, or a fragment or        variant thereof having similar properties;    -   (b) a nucleic acid molecule or polynucleotide sequence encoding        a polypeptide having cellobiohydrolase activity and at least 76%        identity to the full-length amino acid sequence of SEQ ID NO:12,        at least 76% identity to the full-length amino acid sequence of        SEQ ID NO:14, at least 95% identity to the full-length amino        acid sequence of SEQ ID NO:15 or at least 91% identity to the        full-length amino acid sequence of SEQ ID NO:16, or a fragment        or variant thereof having similar properties;    -   c) a nucleic acid molecule comprising the coding sequence of the        polynucleotide sequence depicted as SEQ ID NO:11, SEQ ID NO:13,        SEQ ID NO:9 or SEQ ID NO:10;    -   (d) a nucleic acid molecule comprising the coding sequence of        the polynucleotide sequence contained in DSM 22946, DSM 22945,        DSM 22947 or DSM 23185;    -   (e) a nucleic acid molecule the coding sequence of which differs        from the coding sequence of a nucleic acid molecule of any one        of (c) to (d) due to the degeneracy of the genetic code; and    -   (f) a nucleic acid molecule hybridizing under stringent        conditions to a nucleic acid molecule contained in DSM 22946,        DSM 22945, DSM 22947 or DSM 23185, and encoding a polypeptide        having cellobiohydrolase activity and an amino acid sequence        which shows at least 76% identity to the full-length amino acid        sequence as depicted in SEQ ID NO:12, at least 76% identity to        the full-length amino acid sequence of SEQ ID NO:14, at least        95% identity to the full-length amino acid sequence of SEQ ID        NO:15 or at least 91% identity to the full-length amino acid        sequence of SEQ ID NO:16, or a fragment or variant thereof        having similar properties.

The invention further relates to a recombinant expression vectorcomprising the nucleic acid molecule or polynucleotide sequence of theinvention operably linked to regulatory sequences capable of directingexpression of the gene encoding the CBHII/Cel6A cellobiohydrolase of theinvention and production of said CBHII/Cel6A cellobiohydrolase in asuitable host.

The invention relates also to a host cell comprising the recombinantexpression vector as described above. Preferably, the host is amicrobial host, such as a filamentous fungal host. Preferred hosts areTrichoderma, Aspergillus, Fusarium, Humicola, Chrysosporium, Neurospora,Rhizopus, Penicillium and Mortiriella. More preferably the host isTrichoderma or Aspergillus, most preferably a filamentous fungus T.reesei.

The present invention relates to a process of producing a polypeptide ofthe invention having cellobiohydrolase activity, said process comprisingthe steps of culturing the host cell of the invention and recovering thepolypeptide. Also within the invention is a polypeptide havingcellobiohydrolase activity encoded by the nucleic acid molecule of theinvention and which is obtainable by the process described above.

The invention relates also to a process for obtaining an enzymepreparation comprising the steps of culturing a host cell of theinvention and preparing the whole culture broth, or separating the cellsfrom the spent culture medium and obtaining the supernatant. Within theinvention is also an enzyme preparation obtainable by the processdescribed above. The invention relates also to an enzyme preparation,which comprises the CBHII/Cel6A cellobiohydrolase of the invention.

The enzyme preparation may further comprise other enzymes selected fromthe group: cellobiohydrolase, endoglucanase, beta-glucosidase,beta-glucanase, xyloglucanase, xylanase, beta-xylosidase, mannanase,beta-mannosidase, α-glucuronidase, acetyl xylan esterase,α-arabinofuranosidase, α-galactosidase, pectinase, involving endo- andexo-α-L-arabinases, α-galactosidase, endo- and exo-galactoronase,endopectinlyase, pectate lyase and pectinesterase, phenol esterase,ligninase involving lignin peroxidase, manganese-dependent peroxidase,H₂O₂-generating enzyme and laccase with or without a mediator.

The enzyme preparation may be in the form of whole culture broth orspent culture medium. It may be in the form of liquid, powder orgranulate.

Also within the invention is the use of the CBHII/Cel6A polypeptide orenzyme preparation of the invention for production of biofuel, fordetergents, for treating fibers, for treating food or feed, for pulp andpaper, for beverage or for any applications involving hydrolysis ormodification of cellulosic material.

Particularly, the CBHII/Cel6A polypeptide or enzyme compositioncomprising said polypeptide is useful for production of biofuel.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 schematically shows the expression cassettes used in thetransformation of Trichoderma reesei protoplasts for overproducing therecombinant CBHII/Cel6A proteins. The cbh2/cel6A genes were under thecontrol of T. reesei cbh1/cel7A promoter (p cbh1) and the termination ofthe transcription was ensured by using T. reesei cbh1/cel7A terminatorsequence (t cbh1). The amdS gene was included as a transformationmarker.

FIG. 2 shows determination of pH dependency for the enzyme compositions(100 μg protein in reaction) comprising the recombinant CBHII/Cel6Acellobiohydrolases of Acremonium thermophilum CBS 116240, Melanocarpusalbomyces CBS 685.95; Chaetomium thermophilum CBS 730.95 and Talaromycesemersonii DSM 2432. The hydrolysis was performed on Avicel Ph 101cellulose within a pH range from 3 to 10 at 50° C. for 21 hours. Theformation of reducing sugars was determined bypara-hydroxybenzoic-acidhydrazide (PAHBAH) method (Lever, 1972) using acellobiose standard curve.

FIG. 3 shows determination of thermal stability for the enzymecompositions (100 μg protein in reaction) comprising the recombinantCBHII/Cel6A cellobiohydrolases of Acremonium thermophilum CBS 116240,Melanocarpus albomyces CBS 685.95; Chaetomium thermophilum CBS 730.95and Talaromyces emersonii DSM 2432. The hydrolysis was performed onAvicel Ph 101 cellulose within a temperature range from 40° C. to 80°Cat optimal pH of the enzyme compositions for 21 hours. The formation ofreducing sugars was determined by para-hydroxybenzoic-acidhydrazide(PAHBAH) method (Lever, 1972) using a cellobiose standard curve.

FIG. 4 shows results from hydrolysis of steam exploded hardwood materialperformed with enzyme mixtures comprising the CBHII/Cel6Acellobiohydrolase of the invention. The hardwood substrate washydrolyzed using different enzyme mixtures at a dosage of 5 mg ofprotein per g of total solids both at 55° C. and 37° C. The compositionof the thermophilic enzyme mixture (MIXTURE 2) and the mesophilic enzymemixtures (MIXTURE T. REESEI ENZYMES and MIXTURE ACC), comprising theAt_ALKO4245Cel6A, Ma_ALKO4237_Cel6A or Ct_ALKO4265_Cel6A are describedin more detail in Example 4. Samples from duplicated shake flasks weretaken after a 72 hours hydrolysis time and quantified by HPLC, in whichthe concentration of glucose and xylose were determined. The resultsfrom the substrate blanks, containing buffer instead of the enzymesample, were subtracted from the results obtained with the enzymemixtures. The combined concentration of glucose and xylose is presented.

FIG. 4A shows the hydrolysis results of steam exploded hardwoodperformed at 55° C. with a thermophilic enzyme mixture (MIXTURE 2)supplemented with the At_ALKO4245_Cel6A (MIXTURE 2_AT),Ma_ALKO4237_Cel6A (MIXTURE 2_MA) or Ct_ALKO4265_Cel6A (MIXTURE 2_CT).

FIG. 4B shows the hydrolysis results of steam exploded hardwoodperformed at 37° C. with a mesophilic enzyme mixture (MIXTURE T. REESEIENZYMES) supplemented with the At_ALKO4245_Cel6A (MIXTURE TR_AT),Ma_ALKO4237_Cel6A (MIXTURE TR_MA) or Ct_ALKO4265_Cel6A (MIXTURE TR_CT).

FIG. 4C shows the hydrolysis results of steam exploded hardwoodperformed at 37° C. with a mesophilic enzyme mixture (MIXTURE ACC)supplemented with the At_ALKO4245_Cel6A (MIXTURE ACC_AT),Ma_ALKO4237_Cel6A (MIXTURE ACC_MA) or Ct_ALKO4265_Cel6A (MIXTUREACC_CT).

FIG. 5 shows results from hydrolysis of steam exploded corn cobsmaterial performed with enzyme mixtures comprising a CBHII/Cel6Acellobiohydrolase of the invention. The corn cobs substrate washydrolysed using different enzyme mixtures at a dosage of 5 mg ofprotein per g of total solids at 55° C. The composition of thethermophilic enzyme mixture (MIXTURE 2) comprising the At_ALKO4245_Cel6Apolypeptide of the invention (MIXTURE 2_AT) is described in more detailin Example 5. Samples from duplicated shake flasks were taken after a 72hours hydrolysis time and quantified by HPLC, in which the concentrationof glucose and xylose were determined. The results from the substrateblanks, containing buffer instead of the enzyme sample, were subtractedfrom the results obtained with the enzyme mixtures. The combinedconcentration of glucose and xylose is presented.

SEQUENCE LISTING

SEQ ID NO: 1 Sequence of the oligonucleotide primer CBH_(—)1S

SEQ ID NO: 2 Sequence of the oligonucleotide primer CBH_(—)1AS

SEQ ID NO: 3 Sequence of the oligonucleotide primer CBH_(—)8

SEQ ID NO: 4 Sequence of the oligonucleotide primer CBH_(—)9

SEQ ID NO: 5 Sequence of the oligonucleotide primer Te_CBH_A

SEQ ID NO: 6 Sequence of the oligonucleotide primer Te_CBH_B

SEQ ID NO: 7 Sequence of the PCR fragment obtained from Acremoniumthermophilum ALKO4245 (CBS 116240) using the primers CBH_(—)1S andCBH_(—)1AS.

SEQ ID NO: 8 Sequence of the PCR fragment obtained from Melanocarpusalbomyces ALKO4237 (CBS 685.95) using the primers CBH_(—)1S andCBH_(—)1AS.

SEQ ID NO: 9 The nucleotide sequence of the Chaetomium thermophilumALKO4265 (CBS 730.95) cbh2/cel6A gene.

SEQ ID NO: 10 The nucleotide sequence of the Talaromyces emersoniiRF8069 (DSM 2432) cbh2/cel6A gene.

SEQ ID NO: 11 The nucleotide sequence of the Acremonium thermophilumALKO4245 (CBS 116240) cbh2/cel6A gene.

SEQ ID NO: 12 The deduced amino acid sequence of the Acremoniumthermophilum ALKO4245 (CBS 116240) CBHII/Cel6A.

SEQ ID NO: 13 The nucleotide sequence of the Melanocarpus albomycesALKO4237 (CBS 685.95) cbh2/cel6A gene.

SEQ ID NO: 14 The deduced amino acid sequence of the Melanocarpusalbomyces ALKO4237 (CBS 685.95) CBHII/Cel6A.

SEQ ID NO: 15 The deduced amino acid sequence of the Chaetomiumthermophilum ALKO4265 (CBS 730.95) CBHII/Cel6A.

SEQ ID NO: 16 The deduced amino acid sequence of the Talaromycesemersonii RF8069 (DSM 2432) CBHII/Cel6A.

Depositions

Acremonium thermophilum ALKO4245 was deposited at the CentraalbureauVoor Schimmelcultures at Upsalalaan 8, 3584 CT, Utrecht, the Netherlandson 20 Sep. 2004 and assigned accession number CBS 116240.

Chaetomium thermophilum ALKO4265 was deposited at the CentraalbureauVoor Schimmelcultures at Oosterstraat 1, 3742 SK BAARN, the Netherlandson 8 Nov. 1995 and assigned accession number CBS 730.95. Aftertermination of the current deposit period, samples will be stored underagreements as to make the strain available beyond the enforceable timeof the patent.

Melanocarpus albomyces ALKO4237 was deposited at the Centraalbureau VoorSchimmelcultures at Oosterstraat 1, 3740 AG BAARN, the Netherlands on 11Oct. 1995 and assigned accession number CBS 685.95. After termination ofthe current deposit period, samples will be stored under agreements asto make the strain available beyond the enforceable time of the patent.

The Escherichia coli strain RF8175 including the plasmid pALK2582 wasdeposited at the Deutsche Sammlung von Mikroorganismen and ZellkulturenGmbH (DSMZ), Inhoffenstrasse 7B, D-38124 Braunschweig, Germany on 9 Sep.2009 and assigned accession number DSM 22946.

The E. coli strain RF8174 including the plasmid pALK2581 was depositedat the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH(DSMZ), Inhoffenstrasse 7B, D-38124 Braunschweig, Germany on 9 Sep. 2009and assigned accession number DSM 22945.

The E. coli strain RF8214 including the plasmid pALK2904 was depositedat the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH(DSMZ), Inhoffenstrasse 7B, D-38124 Braunschweig, Germany on 9 Sep. 2009and assigned accession number DSM 22947.

The E. coli strain RF8333 including the plasmid pALK3006 was depositedat the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH(DSMZ), Inhoffenstrasse 7B, D-38124 Braunschweig, Germany on 10 Dec.2009 and assigned accession number DSM 23185.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for treating cellulosic materialwith a CBHII/Cel6A cellobiohydrolase polypeptide or an enzymecomposition comprising said polypeptide. The invention provides alsofungal CBHII/Cel6A cellobiohydrolase polypeptides and enzymecompositions comprising said CBHII/Cel6A polypeptides, whichpolypeptides show broad substrate specificity, and are stable at broadpH and temperature ranges. They have good performance both at moderateand elevated temperatures. Particularly, the CBHII/Cel6Acellobiohydrolase polypeptides are stable for at least 21 hours up to70° C., preferably at a temperature range from 40° C. to 60° C. Thepolypeptides and enzyme compositions comprising said polypeptides areideal in different applications requiring efficient hydrolysis ofcomplex cellulosic or lignocellulosic materials, where cellulose is oneof the major components. The polypeptides and enzyme compositions areuseful, for example in hydrolysis of cellulosic material in order toproduce sugar monomers from the polymeric starting material, which thencan be fermented by microorganisms in the production of biofuel. Thus,the present invention provides alternative CBHII/Cel6Acellobiohydrolases for use in biofuel and other applications. The fungalCBHII/Cel6A cellobiohydrolases can be produced in high-yielding fungalhosts with or without down-stream processing, e.g. separation offermentation broth and mycelia is easy to perform, or in fermentativeorganisms.

“Cellulose” or “cellulosic material” as used herein relates to anymaterial comprising cellulose as a significant component. Cellulose isthe major structural component of higher plants. It provides plant cellswith high tensile strength helping them to resist mechanical stress andosmotic pressure. Cellulose is a β-1,4-glucan composed of linear chainsof glucose residues joined by β-1,4-glycosidic linkages. Cellobiose isthe smallest repeating unit of cellulose. Examples of cellulosicmaterial include textile fibers derived e.g. from cotton, linen, hemp,jute and the manmade cellulosic fibers such as modal, viscose, lyocel.

The term “cellulose” or “cellulosic material” refers also to“lignocellulose” or “lignocellulosic material” comprising cellulose as asignificant component.

In cell walls cellulose is packed in variously oriented sheets, whichare embedded in a matrix of hemicellulose, pectin and/or polymers ofphenol propanol units such as lignin to form “lignocellulosic material”or “lignocellulose”. Lignocellulose is physically hard, dense, andinaccessible. Lignocellulose containing materials include, e.g. plantmaterials such as wood, including hardwood and softwood, hardwood andsoftwoods chips, wood pulp, sawdust and forestry and wood industrialwaste; herbaceous crops, agricultural biomass as cereal straws, sugarbeet pulp, corn fiber, corn stover and cobs, cereal beta-glucans, sugarcane bagasse, stems, leaves, hulls, husks, and the like; waste productsas municipal solid waste, newspaper and waste office paper, waste fiber,paper sludge, milling waste of e.g. grains; dedicated energy crops(e.g., willow, poplar, switchgrass or reed canarygrass, and the like).

Cellulosic and lignocellulosic material is degraded in nature by anumber of various organisms including bacteria and fungi which produceenzymes capable of hydrolyzing carbohydrate polymers. Degradationusually requires the action of many enzymes which typically actsequentially or simultaneously. The biological conversion of celluloseto glucose generally requires three major types of hydrolytic enzymes:(1) Endoglucanases (EG) which cut internal beta-1,4-glycosidic bondsmainly in the amorphous regions of cellulose; (2) Exoglucanases orcellobiohydrolases (CBH) that cut the dissaccharide cellobiose from thereducing or non-reducing end of the crystalline cellulose polymer chain;(3) Beta-1,4-glucosidases (BG) which hydrolyze the cellobiose and othershort cello-oligosaccharides to glucose. Glucose and cellobiose act asend-product inhibitors of the hydrolysis reaction.

“Cellulase” or “cellulolytic enzyme” is an enzyme having “cellulaseactivity” or “cellulolytic activity”, which means that it is capable ofhydrolyzing cellulosic material. One of the most studied cellulolyticenzyme systems is that of the filamentous fungus Trichoderma reeseiwhich is known to exhibit at least 2 CBHs, 8 EGs and 5 BGs.

“Lignocellulolytic enzymes” are enzymes having “lignocellulase activity”or “lignocellulolytic activity”, which means that they are capable ofhydrolyzing lignocellulosic material, such as celluloses,hemicelluloses, or derivatives thereof into smaller saccharides.

The “beta-glucans” are mixed-linkage (1→3),(1→4)-beta-D-glucans. Theyare common in, e.g., cereals.

“Beta-glucanase” as used herein refers to enzymes that can cleave atleast partly the linkages in beta-glucan.

“Cellobiohydrolase” or “CBH” as used herein refers to enzymes thatcleave the 1,4-beta-D-glucosidic linkages of polymers such as cellulosefrom the reducing or non-reducing end and produce mainly cellobiose.They are also called exoglucanases or 1,4-beta-D-glucancellobiohydrolases or cellulose 1,4-beta-cellobiosidases. CBHs have amodular structure consisting of distinct domains, such as a catalyticdomain and an N- or C-terminal cellulose-binding domain (CBD). There arealso cellobiohydrolases in nature which lack CBD. CBHs may also haveadditional domains of unknown function. Different domains are usuallyjoined together by an O-glycosylated linker or hinge peptide rich inglycine, proline, serine or threonine.

By “cellobiohydrolase II” or “CBHII cellobiohydrolase” or “CBHII/Cel6Acellobiohydrolase” or “CBHII/Cel6A cellulase” or “CBHII/Cel6Apolypeptide” or “CBHII/Cel6A enzyme” is in connection of this inventionmeant an 1,4-β-D-glucan cellobiohydrolase enzyme classified as EC3.2.1.91 by the Nomenclature of the International Union of Biochemistryand Molecular Biology (IUBMB). Based on their structural similarities,cellobiohydrolases II or CBHII/Cel6A cellobiohydrolases of the presentinvention are classified into family 6 of glycosyl hydrolases (GH6)having similar amino acid sequences and three-dimensional structures(Henrissat 1991; Henrissat and Bairoch 1993, 1996; Henrissat et al.1998). Because there is a direct relationship between sequence andfolding similarities, such a classification is believed to reflect thestructural features of the enzymes better than their sole substratespecificity, help to reveal the evolutionary relationships between theenzymes, and provide a convenient tool to derive mechanisticinformation.

By the term “cellobiohydrolase activity” or “CBH activity” as used inthe invention is meant hydrolytic activity acting on1,4-beta-D-glycosidic linkages in cellulose, cellotriose, cellotetraoseor in any beta-1,4-linked glucose polymer releasing primarily cellobiosefrom the reducing or non-reducing end of the polymeric chain. Theenzymatic break-down of a glycosidic bond is a stereoselective process,in which the configuration about the anomeric center (C1 carbon) caneither be inverted or retained. Both mechanisms contain a pair ofcarboxylic acid residues disposed on either side of the bond to becleaved. Inverting enzymes use a single-displacement mechanism, whereasthe retaining enzymes involve a double-displacement reaction (Sinnott,1990; Withers and Aebersold, 1995). The stereo chemical course of thehydrolysis is usually determined by proton NMR, in which the α- andβ-anomeric protons give different chemical shifts (Withers et al.,1986).

By the term “cellobiohydrolase II activity” or “CBHII/Cel6A activity” asused in the invention is meant hydrolytic activity acting on1,4-beta-D-glycosidic linkages in cellulose, cellotriose, cellotetraoseor in any beta-1,4-linked glucose polymer releasing primarily cellobiosefrom the non-reducing end of the polymeric chain. The reaction mechanismof CBHII/Cel6A cellobiohydrolases is inverting.

The methods for analyzing cellulase activity are well-known in theliterature and are referred, e.g. by Ghose (1987), Tomme et al. (1988)and van Tilbeurgh et al. (1988). Overall cellulase activity is commonlymeasured as filter paper-degrading activity (FPU). Cellobiohydrolaseactivity may be analyzed using small, soluble cellodextrins and theirchromogenic glycosides, such as 4-methylumbelliferyl-beta-D-glycosides.CBHII/Cel6A cellobiohydrolases can be identified based on the outcome ofthe hydrolysis reaction; CBHII/Cel6A enzymes cannot cleave theheterosidic linkage of small chromogenic oligosaccharides (van Tilbeurghet al., 1988). Cellobiohydrolase activity can be analyzed also onmicrocrystalline cellulose such as Avicel Ph 101, as used in Example 3.The formation of soluble reducing sugars after hydrolysis may bedetermined by para-hydroxybenzoic-acidhydrazide (PAHBAH) method (Lever,1972) using a cellobiose standard curve, the Somogyi-Nelson method(Somogyi, 1952), alkaline ferricyanide method (Robyt and Whelan, 1972),the 2,2′-bichinconinate method (Waffenschmidt and Jaenicke, 1987) or thedinitrosalisylic acid (DNS) method of Miller (1959). Other cellulosicsubstrates include, e.g. Solka floc cellulose or phosphoric acid swollencellulose (Karlsson et al., 2001).

Cellobiohydrolase II can be identified also in a Western or ELISA assayusing polyclonal or monoclonal antibodies raised against the purifiedCBHII/Cel6A protein.

The term “CBHII/Cel6A cellobiohydrolase” thus means cellobiohydrolase IIenzymes which are members of family 6 of glycosyl hydrolases, havingboth a conserved fold and stereochemistry of the hydrolysis reaction.

The term “moderate temperature” or “conventional temperature” in contextof the present invention means temperatures commonly used in cellulosehydrolysis and corresponding to the optimal temperatures or thermalstabilities of the enzymes used in such processes. Thus, the terms referto temperature ranges from 30° C. to 45° C.

The term “elevated temperature” or “high temperature” refers totemperature ranges from 45° C. to 70° C. In short term hydrolysisprocesses the enzymes may be effective even up to 80° C. Enzymes activeor stable at such elevated temperature ranges are also called“thermostable” or “thermophilic” enzymes.

Microorganism strains capable of producing CBHII/Cel6A cellobiohydrolasepolypeptide or CBHII/Cel6A cellobiohydrolase activity can be screened ondifferent substrates. First the chosen strains are cultivated on asuitable medium. After a sufficient amount of an interestingcellobiohydrolase has been produced, the enzyme can be isolated orpurified and its properties can be more thoroughly characterized.Alternatively, genes encoding cellobiohydrolases or CBHII/Cel6Acellobiohydrolases in various organisms can be isolated and the aminoacid sequence encoded by the genes can be compared with the amino acidsequences of the CBHII/Cel6A cellobiohydrolases isolated andcharacterized in Example 1.

The produced cellobiohydrolase enzymes, particularly the CBHII/Cel6Acellobiohydrolase enzymes can be purified by using conventional methodsof enzyme chemistry, such as salt preparation, ultrafiltration, ionexchange chromatography, affinity chromatography, gel filtration andhydrophobic interaction chromatography. Purification can be monitored byprotein determination, enzyme activity assays and by SDS polyacrylamidegel electrophoresis. The enzyme activity and stability of the purifiedenzyme at various temperature and pH values as well as the molecularmass and the isoelectric point can be determined. Alternatively, theproperties of CBHII/Cel6A cellobiohydrolases of the invention may beidentified by producing the enzymes in a recombinant host, and purifyingand characterizing the recombinant CBHII/Cel6A cellobiohydrolasesenzyme. Also, the properties of the recombinant enzyme preparationcomprising the CBHII/Cel6A cellobiohydrolase of the invention as one ofthe major enzyme components may be characterized as described in Example3.

The molecular mass of the purified CBHII/Cel6A cellobiohydrolase can bedetermined by mass spectrometry or on SDS-PAGE according to Laemmli(1970). The molecular mass can also be predicted from the amino acidsequence of the enzyme using the pI/MW tool at ExPASy server(http://expasy.org/tools/pi_tool.html; Gasteiger et al., 2003).

The temperature dependency or thermostability of the CBHII/Cel6Acellobiohydrolase enzyme can be determined in a suitable buffer atdifferent temperatures by using e.g. Avicel cellulose as a substrate asdescribed in Example 3 or by using other substrates and buffer systemsdescribed in the literature. Determination of the pH dependency and pHstability can be carried out in a suitable buffer at different pH valuesby following the activity on a cellulosic substrate.

pI can be determined by isoelectric focusing on an immobilized pHgradient gel composed of polyacrylamide, starch or agarose or byestimating the pI from the amino acid sequence, for example by using thepI/MW tool at ExPASy server (http://expasy.org/tools/pi_tool.html;Gasteiger et al., 2003).

The N-terminus of the purified recombinant CBHII/Cel6A enzyme as well asinternal peptides can be sequenced according to Edman degradationchemistry (Edman and Begg, 1967) or by predicting the cleavage site ofthe secretion signal sequence from the amino acid sequence, e.g. byusing the program SignalP V3.0 (Nielsen et al., 1997; Nielsen and Krogh,1998; Bendtsen et al., 2004) as described in Example 1 or other methodsdescribed in the literature.

The term “full-length” means the form of enzyme translated from thecoding DNA sequence, beginning with the ATG start codon, which encodesthe first methionine in the amino acid sequence and ending at the TGA,TAG or TAA stop codon.

The term “mature” means the form of enzyme which after removal of thesignal sequence (secretion signal peptide or prepeptide) comprises theessential amino acids for enzymatic or catalytic activity. Infilamentous fungi it is the form secreted into the culture medium as aresult, for example of N-terminal processing of the signal sequence andother N-terminal processing, and post-translational glycosylation. Inaddition, the mature form means an enzyme which has been cleaved fromits carrier protein in fusion constructions.

Many of the bacterial and fungal cellobiohydrolases are produced asmodular enzymes (Srisodsuk et al., 1993; Suurnäkki et al., 2000). Inaddition to a catalytic or core domain expressing cellulolytic activitythese enzymes may comprise one or more cellulose binding domains (CBDs),also named as carbohydrate binding domains/modules (CBD/CBM), which canbe located either at the N- or C-terminus of the catalytic domain. CBDshave carbohydrate-binding activity and they mediate the binding of thecellulase enzyme to crystalline cellulose but have little or no effecton cellulase hydrolytic activity of the enzyme on soluble substrates.These two domains are typically connected via a flexible and highlyglycosylated linker or hinge region as is evident from the amino acidsequences of SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16.

“Fragment” as used in the invention means a polypeptide lacking one ormore amino acid residues from the N- and/or C-terminus of thefull-length polypeptide of SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15 orSEQ ID NO:16, such as the mature form of the polypeptide or otherenzymatically active portion of the polypeptide. The fragment still hasthe essential catalytic activity or cellobiohydrolase activity of thefull-length CBHII/Cel6A cellobiohydrolase and substantially similarproperties such as pH and temperature dependence and stability andsubstrate specificity. The polypeptides of the invention disclosed inSEQ ID NO: 12, SEQ ID NO:14, SEQ ID:15 and SEQ ID NO:16 naturallycontain a N-terminal CBD and a linker. These native linker or CBDregions may be replaced by, e.g. a CBD from a Trichoderma or Chaetomiumspecies. The CBHII/Cel6A enzymes of the invention may be used in theapplications also without a signal sequence and/or CBD or the signalsequence and/or CBD may derive from different enzymes of the abovemicroorganisms or different microorganism or be synthetically orrecombinantly incorporated to the catalytic domain of the above enzymes.

The term “identity” as used herein means the identity between two aminoacid sequences compared to each other within the corresponding sequenceregion having approximately the same amount of amino acids. For example,the identity of a full-length or a mature sequence of the two amino acidsequences may be compared. Also, the identity of a full-length or amature sequence lacking the N-terminal CBD of the two amino acidsequences may be compared. Thus, comparison of e.g. the CBHII/Cel6Asequences including CBD and/or signal sequences with sequences lackingthose elements is not within the context of the invention. The identityof the full-length sequences may be measured by using, for exampleClustalW alignment (e.g. in www.ebi.ac.uk/Tools/Clustalw) with a matrixas follows: BLOSUM, Gap open:10, Gap extension: 0.5, or using a programof Clone Manager (version 9) (Scientific and Educational Software, Cary,USA), including the functions “Compare Two Sequences/Global/Comparesequences as amino acids/BLOSUM62 scoring matrix as described in Example1.

The amino acid sequences of the two molecules to be compared may differin one or more positions, which however do not alter the biologicalfunction or structure of the molecules. Such “variants” may occurnaturally because of different host organisms or mutations in the aminoacid sequence, e.g. as an allelic variant within the same strain,species or genus, or they may be achieved by specific mutagenesis. Theymay comprise amino acid substitutions, deletions, combinations orinsertions of one or more positions in the amino acid sequence, but theystill function in a substantially similar manner to the enzymes definedin SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15 or SEQ ID NO:16, i.e. theycomprise a variant having cellulolytic activity.

The present invention relates to a method for treating cellulosicmaterial, including also lignocellulosic material with a CBHII/Cel6Apolypeptide or an enzyme composition comprising said polypeptide or asin a consolidated bioprocess, the cellulosic material may be treatedwith a fermentative microorganism producing said polypeptide, whereinthe CBHII/Cel6A polypeptide exhibits cellobiohydrolase activity andcomprises an amino acid sequence having at least 76% identity to thefull-length polypeptide of SEQ ID NO: 12, at least 76% identity to thefull-length polypeptide of SEQ ID NO:14, at least 95% identity to thefull-length polypeptide of SEQ ID NO:15, or at least 91% identity to thefull-length polypeptide of SEQ ID NO:16, or a fragment or variantthereof having similar properties. Said enzyme is capable in hydrolyzingcellulosic material, including lignocellulose at moderate to elevatedtemperatures. Said method comprises the following steps: i) productionof a CBHII/Cel6A polypeptide of the invention or an enzyme compositioncomprising said polypeptide or a fermentative microorganism producingsaid polypeptide; ii) reacting the cellulosic material with theCBHII/Cel6A polypeptide of the invention or the enzyme compositioncomprising said polypeptide or the fermentative microorganism producingsaid polypeptide; and iii) obtaining partially or fully hydrolyzedcellulosic material, including hydrolyzed lignocellulosic material.

CBHII/Cel6A cellobiohydrolase enzymes useful for treating or hydrolyzingcellulosic material are “obtainable from” any organism including plants.Preferably CBHII/Cel6A enzymes originate from microorganisms, e.g.bacteria or fungi. The bacteria may be, for example from a genusselected from Bacillus, Azospirillum, Streptomyces and Pseudomonas.

More preferably the enzyme originates from fungi (including filamentousfungi and yeasts), for example from a genus selected from the groupconsisting of yeasts Schizosaccharomyces, Kluyveromyces, Pichia,Saccharomyces, Candida and Yarrowia or filamentous fungi Achaetomium,Acremonium, Aspergillus, Aureobasidium, Botrytis, Chaetomium,Chrysosporium, Cryptococcus, Collybia, Fomes, Fusarium, Humicola,Hypocrea, Lentinus, Magnaporthea, Melanocarpus, Mucor, Myceliophthora,Myriococcum, Neocallimastix, Neurospora, Paecilomyces, Penicillium,Phanerochaete, Phlebia, Pleurotus, Podospora, Polyporus, Pycnoporus,Rhizoctonia, Schizophyllum, Scytalidium, Talaromyces, Thermoascus,Thielavia, Trametes and Trichoderma. Preferably the CBHII/Cel6A enzymesderive from Acremonium thermophilum, Melanocarpus albomyces, Chaetomiumthermophilum or Talaromyces emersonii.

According to a preferred embodiment of the invention the enzymes areobtainable from a filamentous fungal strain ALKO4245 deposited as CBS116240 and presently classified as Acremonium thermophilium,Melanocarpus albomyces strain ALKO4237 deposited as CBS 685.95,Talaromyces emersonii strain DSM 2432 (in applicant's culture collectionunder the number RF8069) or Chaetomium thermophilum strain ALKO4265deposited as CBS 730.95.

The CBHII/Cel6A cellobiohydrolases of the present invention are markedas At_ALKO4245_Cel6A, Ma_ALKO4237_Cel6A, Ct_ALKO4265_Cel6A andTe_RF8069_Cel6A, being the CBHII/Cel6A cellobiohydrolases originatingfrom the strains Acremonium thermophilum CBS 116240, Melanocarpusalbomyces CBS 685.95, Chaetomium thermophilum CBS 730.95 or Talaromycesemersonii DSM 2432 and members of family 6 of glycoside hydrolases.

TABLE 1 The CBHII/Cel6A cellobiohydrolases of the invention Cellobio-Obtainable nucleic acid amino acid hydrolase II Gene from SEQ ID NO: SEQID NO: At_ALKO4245_Cel6A At_ALKO4245_cel6A Acremonium 11 12 thermophilumMa_ALKO4237_Cel6A Ma_ALKO4237_cel6A Melanocarpus 13 14 albomycesCt_ALKO4265_Cel6A Ct_ALKO4265_cel6A Chaetomium 9 15 thermophilumTe_RF8069_Cel6A Te_RF8069_cel6A Talaromyces 10 16 emersonii

According to a preferred embodiment of the invention the fungalCBHII/Cel6A cellobiohydrolase enzyme useful in the method is apolypeptide having cellobiohydrolase activity and comprising the enzymeof At_ALKO4245_Cel6A having the full-length amino acid sequence of SEQID NO: 12 or an amino acid sequence having at least 76% identity to theamino acid sequence SEQ ID NO: 12. Preferred enzymes show at least 78%,80% or 82%, preferably at least 84%, 86% or 88%, more preferably atleast 90%, even more preferably at least 92% identity. Still morepreferably the amino acid sequences show at least 94% or at least 96% or97%, more preferably at least 98%, most preferably 99% identity to theamino acid sequence of SEQ ID NO: 12. The identities of the two enzymesare compared within the corresponding sequence regions, i.e. within thefull-length region of the CBHII/Cel6A cellobiohydrolase.

Another preferred embodiment of the invention is a fungal CBHII/Cel6Acellobiohydrolase enzyme useful in the method having cellobiohydrolaseactivity and comprising the enzyme of Ma_ALKO4237_Cel6A having thefull-length amino acid sequence of SEQ ID NO:14 or an amino acidsequence having at least 76% identity to the amino acid sequence SEQ IDNO:14. Preferred enzymes show at least 78%, 80% or 82%, preferably atleast 84%, 86% or 88%, more preferably at least 90%, even morepreferably at least 92% identity. Still more preferably the amino acidsequences show at least 94% or at least 96% or 97%, more preferably atleast 98%, most preferably 99% identity to the amino acid sequence ofSEQ ID NO: 14. The identities of the two enzymes are compared within thecorresponding sequence regions, i.e. within the full-length region ofthe CBHII/Cel6A cellobiohydrolase.

A further preferred embodiment of the invention is a fungal CBHII/Cel6Acellobiohydrolase enzyme useful in the method having cellobiohydrolaseactivity and comprising the enzyme of Ct_ALKO4265_Cel6A having thefull-length amino acid sequence of SEQ ID NO:15 or an amino acidsequence having at least 95% identity to the amino acid sequence SEQ IDNO:15. Preferred enzymes show at least 96%, preferably at least 97%,more preferably at least 98%, most preferably at least 99% identity tothe amino acid sequence of SEQ ID NO: 15. The identities of the twoenzymes are compared within the corresponding sequence regions, i.e.within the full-length region of the CBHII/Cel6A cellobiohydrolase.

Still further preferred embodiment of the invention is a fungalCBHII/Cel6A cellobiohydrolase enzyme useful in the method havingcellobiohydrolase activity and to comprising the enzyme ofTe_RF8069_Cel6A having the full-length amino acid sequence of SEQ IDNO:16 or an amino acid sequence having at least 91% identity to theamino acid sequence SEQ ID NO:16. Preferred enzymes show at least 92%,more preferably at least 93%, even more preferably at least 94%identity. Still more preferably the amino acid sequences show at least95% or at least 96% or 97%, more preferably at least 98%, mostpreferably 99% identity to the amino acid sequence of SEQ ID NO: 16. Theidentities of the two enzymes are compared within the correspondingsequence regions, i.e. within the full-length region of the CBHII/Cel6Acellobiohydrolase.

The fungal CBHII/Cel6A cellobiohydrolases of the invention are active orstable over a broad pH range of at least pH 3 to pH 10 and morepreferably at a pH range of at least pH 3 to pH 7 when assayed at 50° C.for 21 hours using Avicel cellulose as a substrate, as described inExample 3.

In particular, the At_ALKO4245_Cel6A is active between pH 3 and pH 7,preferably between pH 4 and pH 6, and more preferably between pH 4 andpH 5. The maximum activity of At_ALKO4245_Cel6A is at pH 5 when assayedat 50° C. for 21 hours using Avicel cellulose as a substrate.

The Ma_ALKO4237_Cel6A is active at a pH range between pH 3 and pH 10,preferably between pH 4 and pH 8, more preferably between pH 4 and pH 7,still more preferably between pH 4 and pH 6, and even more preferablybetween pH 4 and pH 5. The maximum activity of Ma_ALKO4237_Cel6A is atpH 4 when assayed at 50° C. for 21 hours using Avicel cellulose as asubstrate.

The Ct_ALKO4265_Cel6A is active at a pH range between pH 3 and pH 10,preferably between pH 3 and pH 8, more preferably between pH 3 and pH 7,still more preferably between pH 4 and pH 7, and even more preferablybetween pH 4 and pH 6. The maximum activity of Ct_ALKO4265_Cel6A is atpH 5 when assayed at 50° C. for 21 hours using Avicel cellulose as asubstrate.

The Te_RF8089_Cel6A is active at a pH range between pH 3 and pH 7,preferably between pH 3 and pH 6, and more preferably between pH 4 andpH 6. The maximum activity of Te_RF8089_Cel6A is at pH 4 when assayed at50° C. for 21 hours using Avicel cellulose as a substrate.

The enzymes of the invention are effective in degrading cellulosic orlignocellulosic material at a broad temperature range. The CBHII/Cel6Acellobiohydrolases of the invention are active or stable for up to 21hours in a temperature range between 40° C. and 70° C. when assayed atthe optimum pH of the enzymes using Avicel cellulose as a substrate, asdescribed in Example 3.

The At_ALKO4245_Cel6A cellobiohydrolase is active between 40° C. and 70°C., preferably between 40° C. and 60° C., more preferably between 50° C.and 60° C. At optimal pH the maximum activity of At_ALKO4245_Cel6A is at60° C. when using a 21 hours incubation time and Avicel cellulose as asubstrate.

The Ma_ALKO4237_Cel6A cellobiohydrolase is active in a temperature rangebetween 40° C. and 60° C. The enzyme shows maximum activity at 50° C.when incubated for 21 hours at the optimal pH using Avicel cellulose asa substrate. The Ct_ALKO4265_Cel6A cellobiohydrolase is active in atemperature range between 40° C. and 70° C., preferably in the range of50° C. to 60° C. At the optimal pH the maximum activity is at 60° C.when using a 21 hours incubation time and Avicel cellulose as asubstrate. The Te_RF8089_Cel6A cellobiohydrolase is active between 40°C. and 70° C., preferably between 40° C. and 60° C., more preferablybetween 50° C. and 60° C. The maximum activity is at 60° C. whenincubated for 21 hours at the optimal pH using Avicel cellulose as asubstrate.

The cellulolytic enzymes presently in use in hydrolysis of cellulosicmaterial and production of fermentable sugars for, e.g. bioethanolapplications derive mainly from the well-studied microorganisms, such asthe filamentous fungus Trichoderma reesei (e.g. the Accellerase® productline, Genencor Int., Inc., US). The cellulolytic enzymes areconventionally used at temperatures in the range of 30° C. to 45° C. TheCBHII/Cel6A cellobiohydrolases of the present invention are efficient atthese temperatures too, but in addition they work extremely well even attemperatures up to 70° C., such as between 40° C. and 70° C., e.g.between 40° C. and 70° C. or between 40° C. and 60° C. or between 50° C.and 60° C., as shown in Example 3. For short hydrolysis times enzymecompositions may be functional up to 80° C. For total hydrolysis longerincubation times are required and, therefore, lower temperatures arenormally used. This makes the CBHII/Cel6A cellobiohydrolases of theinvention extremely well suited for varying cellulosic substratehydrolysis processes carried out both at conventional or moderatetemperatures and at elevated temperatures.

In Examples 4 and 5, experiments performed on various cellulosicmaterials, such as hardwood and corn cobs are described. From FIG. 4 itis evident that the performance of the enzyme mixtures supplemented withthe fungal CBHII/Cel6A cellobiohydrolases At_ALKO4245_Cel6A,Ma_ALKO4237_Cel6A or Ct_ALKO4265_Cel6A in hydrolysis of steam explodedhardwood is by far better than the performance of the enzyme mixtureswithout such supplementation. In the experiments performed at 55° C.(FIG. 4A), the amount of sugars released from the hardwood substrate wasfound to increase 12%, 14% and 26% by supplementing withMa_ALKO4237_Cel6A, Ct_ALKO4265_Cel6A or At_ALKO4245_Cel6A enzymes in theMIXTURE 2, respectively. Acremonium thermophilum ALKO4245 enzyme wasfound to be best-performing CBHII/Cel6A herein studied.At_ALKO4245_Cel6A shows increased hydrolysis also at 37° C. added eitherin the state-of-the-art Trichoderma mixture (MIXTURE T. REESEI ENZYMES)(FIG. 4B) or in the commercial product (MIXTURE ACC) (FIG. 4C).

Similar results were obtained also on steam exploded corn cobs (FIG. 5).The thermophilic enzyme MIXTURE 2 was supplemented with theAt_ALKO4245_Cel6A enzyme of the invention. The combined amount ofglucose and xylose after 72 hours hydrolysis was remarkably higher thanwithout such supplementation.

According to one preferred embodiment of the invention the method fortreating cellulosic and lignocellulosic material, such as thelignocellulosic material including hemicellulose, pectin and lignininvolves use of one or more of the CBHII/Cel6A cellobiohydrolases of theinvention as an “enzyme composition” or “enzyme preparation” comprisingat least one further enzyme capable of hydrolyzing said material. Theadditional enzymes may be selected from a group of cellobiohydrolase,endoglucanase, beta-glucosidase, beta-glucanase, xyloglucanase,xylanase, beta-xylosidase, mannanase, beta-mannosidase, α-glucuronidase,acetyl xylan esterase, α-arabinofuranosidase, α-galactosidase,pectinase, involving endo- and exo-α-L-arabinases, α-galactosidase,endo- and exo-galactoronase, endopectinlyase, pectinesterase, pectatelyase, phenol esterase, ligninase involving lignin peroxidase,manganese-dependent peroxidase, H₂O₂-generating enzyme and laccase withor without a mediator.

The enzyme preparation or composition comprises at least one of theenzymes defined above. It may contain the enzymes in at least partiallypurified and isolated form. It may even essentially consist of thedesired enzyme or enzymes. Alternatively the preparation may be a spentculture medium or filtrate containing one or more of the desiredenzymes. Preferably the enzyme preparation is spent culture medium.“Spent culture medium” refers to the culture medium of the hostcomprising the produced enzymes. Preferably the host cells are separatedfrom said medium after the production. The enzyme preparation orcomposition may also be a “whole culture broth” obtained, optionallyafter killing the production host(s) or microorganism(s) without anyfurther down-stream processing or purification of the desiredcellulolytic enzyme(s). In the “consolidated bioprocess” the enzymecomposition or at least some of the enzymes of the enzyme compositionmay be produced by the fermentative microorganism.

“Isolated polypeptide” in the present context may simply mean that thecells and cell debris have been removed from the culture mediumcontaining the polypeptide. Conveniently the polypeptides are isolated,e.g. by adding anionic and/or cationic polymers to the spent culturemedium to enhance precipitation of cells, cell debris and some enzymesthat have unwanted side activities. The medium is then filtrated usingan inorganic filtering agent and a filter to remove the precipitantsformed. After this the filtrate is further processed using asemi-permeable membrane to remove excess of salts, sugars and metabolicproducts.

In addition to the enzymatic activity, the preparation may containadditives, such as mediators, stabilizers, buffers, preservatives,surfactants and/or culture medium components. Preferred additives aresuch, which are commonly used in enzyme preparations intended for aparticular application. The enzyme preparation may be in the form ofliquid, powder or granulate.

According to one embodiment of the invention the enzyme preparationcomprises a mixture of CBHs, EGs and BGs, optionally together withhemicellulose degrading enzymes in combination with the CBHII/Cel6Acellobiohydrolases of the invention. Different enzyme mixtures andcombinations may be used to suit different substrate materials andprocess conditions. For example if the degradation process is to becarried out at a high temperature, thermostable enzymes are chosen.

“Hemicellulose” is a heterogeneous group of carbohydrate polymerscontaining mainly different glucans, xylans and mannans. Hemicelluloseconsists of a linear backbone with β-1,4-linked residues substitutedwith short side chains usually containing acetyl acid, 4-O-glucuronicacid, L-arabinose and galactosyl groups. Hemicellulose may be chemicallycross-linked to lignin and cellulose. “Xylan degrading enzymes” or“xylanases” include both exohydrolytic and endohydrolytic enzymes suchas endo-1,4-beta-D-xylanase (EC 3.2.1.8) or exo-1,4-beta-D-xylosidase(EC 3.2.1.37), which break down hemicellulose to xylose. Gluco- andgalactomannans are hydrolyzed by endo-1,4-beta-mannanases (EC3.2.1.78)and beta-mannosidase (EC 3.2.1.25) to yield beta-D-mannose. Enzymescapable of removing side chain substituents include α-glucuronidases,acetyl xylan esterases, α-arabinofuranosidases and α-galactosidaseswhich act cooperatively with the backbone degrading enzymes (Biely etal. 1997; Sundberg and Poutanen, 1991; Stålbrand et al., 1995).

Enzymes involved in degradation of the “pectin” involve endo- andexo-α-L-arabinases and α-galactosidase, endo- and exo-galactoronases,endopectinlyases and pectinesterases (Del Cañizo et al., 1994).

“Lignin” is a complex cross-linked polymer of variously substitutedp-hydroxyphenylpropane units. Its hydrolysis involves ligninperoxidases, phenol esterases, manganese-dependent peroxidases,H₂O₂-generating enzymes and laccases (Cullen and Kersten, 2004).

The enzymes of the enzyme composition may be added to thelignocellulosic material either simultaneously or sequentially.

According to one preferred embodiment of the invention, the method isapplicable on various cellulose, lignocellulose and beta-glucancontaining materials, such as plant materials, e.g. wood, includinghardwood and softwood, hardwood and softwoods chips, wood pulp, sawdustand forestry and wood industrial waste; herbaceous crops, agriculturalbiomass as cereal straws, sugar beet pulp, corn fiber, corn stover andcobs, cereal beta-glucans, sugar cane bagasse, stems, leaves, hulls,husks, and the like; waste products as municipal solid waste, newspaperand waste office paper, waste fiber, paper sludge, milling waste of e.g.grains; dedicated energy crops (e.g., willow, poplar, switchgrass orreed canarygrass, and the like).

According to one embodiment of the invention, the method is applicablefor production of biofuels, such as ethanol, propanol and butanol andalike from cellulosic material.

Within the context of the invention is a method, wherein the CBHII/Cel6Apolypeptide used in the hydrolysis of the cellulosic material derivesfrom Acremonium thermophilum CBS 116240 strain and has the amino acidsequence of SEQ ID NO:12 or at least 78%, 80%, 82%, 84%, 86%, 88%, 90%,92%, 94%, 96%, 97%, 98% or 99% identity with the amino acid sequence ofSEQ ID NO:12, or a fragment or variant thereof having cellobiohydrolaseactivity.

The present invention relates also to a fungal CBHII/Cel6A polypeptidehaving cellobiohydrolase activity and comprising an amino acid sequencehaving at least 76% identity to the full-length polypeptide of SEQ IDNO: 12, at least 76% identity to the full-length polypeptide of SEQ IDNO:14, at least 95% identity to the full-length polypeptide of SEQ IDNO:15, or at least 91% identity to the full-length polypeptide of SEQ IDNO:16, or a fragment or variant thereof having similar properties.

The CBHII/Cel6A polypeptide may be obtainable from the filamentousfungal genus Acremonium, Melanocarpus, Chaetomium or Talaromyces.Preferable species include Acremonium thermophilum, Melanocarpusalbomyces, Chaetomium thermophilum or Talaromyces emersonii.

According to a preferred embodiment of the invention the enzymes areobtainable from a filamentous fungal strain ALKO4245 deposited as CBS116240 presently classified as Acremonium thermophilium, Melanocarpusalbomyces strain ALKO4237 deposited as CBS 685.95, Chaetomiumthermophilum strain ALKO4265 deposited as CBS 730.95 or Talaromycesemersonii strain DSM 2432 (in the applicant's culture collection undernumber RF8069).

According to a preferred embodiment of the invention the fungalCBHII/Cel6A cellobiohydrolase enzyme is a polypeptide havingcellobiohydrolase activity and comprising the enzyme ofAt_ALKO4245_Cel6A having the full-length amino acid sequence of SEQ IDNO: 12.

Another preferred embodiment of the invention is a fungal CBHII/Cel6Acellobiohydrolase enzyme having cellobiohydrolase activity andcomprising the enzyme of Ma_ALKO4237_Cel6A with the full-length aminoacid sequence of SEQ ID NO: 14.

A further preferred embodiment of the invention is a fungal CBHII/Cel6Acellobiohydrolase enzyme having cellobiohydrolase activity andcomprising the enzyme of Ct_ALKO4265_Cel6A with the full-length aminoacid sequence of SEQ ID NO: 15.

Still further preferred embodiment of the invention is a fungalCBHII/Cel6A cellobiohydrolase enzyme having cellobiohydrolase activityand comprising the enzyme of Te_RF8069_Cel6A with the full-length aminoacid sequence of SEQ ID NO: 16.

According to one preferred embodiment of the invention, the CBHII/Cel6Acellobiohydrolase enzyme is capable in hydrolyzing cellulosic materialat moderate to elevated temperatures. The CBHII/Cel6A cellobiohydrolasesof the invention are active or stable even at temperatures up to 70° C.,such as between 40° C. and 70° C., e.g. between 40° C. and 60° C. orbetween 50° C. and 60° C., when assayed at the optimum pH of the enzymesusing Avicel Ph101 cellulose as a substrate, as described in Example 3.For short hydrolysis times enzyme compositions may be functional up to80° C. For total hydrolysis longer incubation times are required and,therefore, lower temperatures are normally used. This makes theCBHII/Cel6A cellobiohydrolases of the invention extremely well suitedfor varying cellulosic substrate hydrolysis processes carried out bothat conventional or moderate temperatures and at elevated temperaturesrequiring thermostability of the enzymes.

According to a preferred embodiment of the invention the fungalCBHII/Cel6A is encoded by an isolated nucleic acid molecule whichcomprises a polynucleotide sequence, which encodes a polypeptidecomprising the amino acid sequence characterized in SEQ ID NO:12, SEQ IDNO:14, SEQ ID NO:15 or SEQ ID NO:16. Thus, within the scope of theinvention is CBHII/Cel6A cellobiohydrolase enzyme or polypeptidecomprising the amino acid sequence of the full-length form of theAt_ALKO4245_Cel6A enzyme characterized in SEQ ID NO: 12,Ma_ALKO4237_Cel6A enzyme characterized in SEQ ID NO: 14, Ct_ALKO4265characterized in SEQ ID NO:15 or Te_RF8069_Cel6A characterized in SEQ IDNO:16.

Further, within the scope of the present invention are polypeptidesencoded by nucleic acid molecules encoding a CBHII/Cel6A polypeptidehaving cellobiohydrolase activity and at least 76% identity to thefull-length amino acid sequence of SEQ ID NO: 12, at least 76% identityto the full-length amino acid sequence of SEQ ID NO: 14, at least 95%identity to the full-length amino acid sequence of SEQ ID NO:15 or atleast 91% to the full-length amino acid sequence of SEQ ID NO:16. Theidentities of the two enzymes are compared within the correspondingsequence regions, i.e. within the full-length region of the CBHII/Cel6Apolypeptide.

Within the scope of the invention is a polypeptide sequence, which isencoded by a nucleic acid molecule coding for a fragment of thepolypeptide, which polypeptide fragment lacks one or more amino acidresidues from the N- and/or C-terminus of the full-length polypeptide ofSEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO:15 or SEQ ID NO:16. The fragmentstill has the essential catalytic activity or cellobiohydrolase activityof the full-length CBHII/Cel6A cellobiohydrolase and substantiallysimilar properties such as pH and temperature dependence and substratespecificity. The fragment may, for example be an enzyme which lacks thesecretion signal sequence or carbohydrate binding domain or CBD.

Also included are natural or synthetic variants of the CBHII/Cel6Acellobiohydrolases, which have properties similar to the polypeptidesdefined in SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO:15 or SEQ ID NO:16.The variation may result from deletion, substitution, insertion,addition or combination of one or more positions in the amino acidsequence.

One preferred embodiment of the invention is a CBHII/Cel6Acellobiohydrolase which is encoded by an isolated nucleic acid moleculecomprising a polynucleotide sequence included in SEQ ID NO:11, SEQ IDNO:13, SEQ ID NO:9 or SEQ ID NO:10 or a subsequence thereof.

Within the context of the invention is a CBHII/Cel6A cellobiohydrolase,which is encoded by a nucleic acid molecule or polynucleotide sequencehybridizing under stringent conditions to a polynucleotide sequence or asubsequence thereof included in SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:9or SEQ ID NO:10. Also, a preferred embodiment is a polypeptide encodedby a nucleic acid molecule or polynucleotide sequence hybridizing with aprobe prepared using PCR, such as the PCR fragment included in SEQ IDNO:7 or SEQ ID NO:8.

Standard molecular biology methods can be used in isolation of cDNA or agenomic DNA of the host organism, e.g. the methods described in themolecular biology handbooks, such as Sambrook and Russell, 2001. ThecDNA or a genomic gene encoding the CBHII/Cel6A cellobiohydrolase of theinvention may be isolated using DNA probes, which have been preparedbased on the amino acid sequence of N-terminal or tryptic peptides ofthe purified CBHII/Cel6A enzyme. Alternatively, the probe may bedesigned based on the known nucleotide or amino acid sequences ofhomologous cellobiohydrolases. The CBHII/Cel6A clones may also bescreened based on activity on plates containing a specific substrate forthe enzyme or by using antibodies specific for a CBHII/Ce6Acellobiohydrolase.

Hybridization with a DNA probe, such as for example SEQ ID NO: 7consisting of more than 100-200 nucleotides, is usually performed at“high stringency” conditions, i.e. hybridization at a temperature, whichis 20-25° C. below the calculated melting temperature (Tm) of a perfecthybrid, the Tm calculated according to Bolton and McCarthy (1962) andposthybridization washes in low salt concentration. Usuallyprehybridization and hybridization are performed at least at 65° C. in6×SSC (or 6×SSPE), 5×Denhardt's reagent, 0.5% (w/v) SDS, 100 μg/mldenatured, fragmented salmon sperm DNA. Addition of 50% formamide lowersthe prehybridization and hybridization temperatures to 42° C. Highstringency washes are performed in low salt concentration, e.g. in2×SSC-0.1% SDS (w/v) at RT, and finally in 0.1×SSC-0.1% SDS (w/v) atleast at 65° C., e.g. at 68° C.

In the present invention the At_ALKO4245_cel6A, Ma_ALKO4237_cel6A,Ct_ALKO4265_cel6A and Te_RF8069_cel6A genes were isolated with a probeprepared by PCR using stringent hybridization conditions as described inExample 1. Genomic cbh2/cel6A genes were isolated by usingoligonucleotide primers designed based on the published nucleotidesequences or using degenerate oligonucleotide primers planned based onthe alignment of the previously known amino acid sequences ofCBHII/Cel6A proteins.

According to one preferred embodiment of the invention the fungalCBHII/Cel6A cellobiohydrolase enzyme is encoded by an isolated nucleicacid molecule, which comprises the nucleotide sequence of SEQ ID NO:11encoding the full-length form of the At_ALKO4245_Cel6A enzyme of SEQ IDNO:12. Another preferred embodiment of the invention is a fungalCBHII/Cel6A cellobiohydrolase encoded by an isolated nucleic acidmolecule comprising the nucleotide sequence of SEQ ID NO:13, whichencodes the full-length form of the Ma_ALKO4237_Cel6A enzyme having theamino acid sequence of SEQ ID NO:14. Another preferred embodiment of theinvention is a fungal CBHII/Cel6A cellobiohydrolase encoded by anisolated nucleic acid molecule comprising the nucleotide sequence of SEQID NO:9, which encodes the full-length form of the Ct_ALKO4265_Cel6Aenzyme having the amino acid sequence of SEQ ID NO:15. Further preferredembodiment of the invention is a fungal CBHII/Cel6A cellobiohydrolaseencoded by an isolated nucleic acid molecule comprising the nucleotidesequence of SEQ ID NO:10, which encodes the full-length form of theTe_RF8069_Cel6A enzyme having the amino acid sequence of SEQ ID NO:16.

According to another preferred embodiment of the invention the fungalCBHII/Cel6A cellobiohydrolase is encoded by the polynucleotide sequenceincluded in plasmid pALK2582 deposited in Escherichia coli RF8175 underaccession number DSM 22946, plasmid pALK2581 deposited in Escherichiacoli RF8174 under accession number DSM 22945, plasmid pALK2904 depositedin Escherichia coli RF8214 under accession number DSM 22947, or plasmidpALK3006 deposited in Escherichia coli RF8333 under accession number DSM23185.

One embodiment of the invention is the CBHII/Cel6A cellobiohydrolaseproduced from a recombinant expression vector comprising the nucleicacid molecule, which encodes the fungal CBHII/Cel6A cellobiohydrolase ascharacterized above, operably linked to regulatory sequences capable ofdirecting the expression of a gene encoding said CBHII/Cel6Acellobiohydrolase enzyme in a suitable host. Construction of saidrecombinant expression vector and use of said vector is described inmore detail in Example 2.

Suitable hosts for production of the fungal CBHII/Cel6Acellobiohydrolase are homologous or heterologous hosts, such as themicrobial hosts including bacteria, yeasts and fungi. Filamentous fungi,such as Trichoderma, Aspergillus, Fusarium, Humicola, Chrysosporium,Neurospora, Rhizopus, Penicillium and Mortiriella, are preferredproduction hosts due to the ease of down-stream processing and recoveryof the enzyme product. Suitable hosts include species such as T. reesei,A. niger, A oryzae, A. sojae, A. awamori or A. japonicus type ofstrains, F. venenatum or F. oxysporum, H. insolens or H. lanuginosa, N.crassa and C. lucknowense, some of which are listed as enzyme productionhost organisms in e.g. AMFEP 2007 list of commercial enzymes(http://www.amfep.org/list.html). More preferably, the enzyme isproduced in a filamentous fungal host of the genus Trichoderma orAspergillus, such as T. reesei, or A. niger, A. oryzae or A. awamori.According the most preferred embodiment of the invention the fungalCBHII/Cel6A cellobiohydrolase enzyme is produced in T. reesei.

According to the preferred embodiment of the invention the CBHII/Cel6Apolypeptide is At_ALKO4245_Cel6A deriving from Acremonium thermophilumCBS 116240 and having the amino acid sequence of SEQ ID NO: 12.

The present invention relates also to an isolated nucleic acid moleculecomprising a polynucleotide sequence encoding the fungal CBHII/Cel6Acellobiohydrolase selected from the group consisting of:

-   -   (a) a nucleic acid molecule or polynucleotide sequence encoding        a polypeptide having cellobiohydrolase activity and comprising        the full-length amino acid sequence as depicted in SEQ ID NO:12,        SEQ ID NO:14, SEQ ID NO:15 or SEQ ID NO:16, or a fragment or        variant thereof having similar properties;    -   (b) a nucleic acid molecule or polynucleotide sequence encoding        a polypeptide having cellobiohydrolase activity and at least 76%        identity to the full-length amino acid sequence of SEQ ID NO:12,        at least 76% identity to the full-length amino acid sequence of        SEQ ID NO:14, at least 95% identity to the full-length amino        acid sequence of SEQ ID NO:15 or at least 91% identity to the        full-length amino acid sequence of SEQ ID NO:16, or a fragment        or variant thereof having similar properties;    -   (c) a nucleic acid molecule comprising the coding sequence of        the nucleotide sequence as depicted in SEQ ID NO: 11, SEQ ID        NO:13, SEQ ID NO:9 or SEQ ID NO:10;    -   (d) a nucleic acid molecule comprising the coding sequence of        the polynucleotide sequence contained in DSM 22946, DSM 22945,        DSM 22947 or DSM 23185;    -   (e) a nucleic acid molecule the coding sequence of which differs        from the coding sequence of a nucleic acid molecule of any one        of (c) to (d) due to the degeneracy of the genetic code; and    -   (f) a nucleic acid molecule hybridizing under stringent        conditions to a nucleic acid molecule contained in DSM 22946,        DSM 22945, DSM 22947 or DSM 23185 or a subsequence thereof, and        encoding a polypeptide having cellobiohydrolase activity and an        amino acid sequence which shows at least 76% identity to the        full-length amino acid sequence as depicted in SEQ ID NO:12, at        least 76% to the full-length amino acid sequence of SEQ ID        NO:14, at least 95% to the full-length amino acid sequence of        SEQ ID NO:15 or at least 91% to the full-length amino acid        sequence of SEQ ID NO:16.

The nucleic acid molecule of the invention may be RNA or DNA, whereinthe DNA may constitute of the genomic DNA or cDNA.

Standard molecular biology methods can be used in isolation and enzymetreatments of the polynucleotide sequence encoding the fungalCBHII/Cel6A cellobiohydrolase of the invention, including isolation ofgenomic and plasmid DNA, digestion of DNA to produce DNA fragments,sequencing, E. coli transformations etc. The basic methods are describedin the standard molecular biology handbooks, e.g. Sambrook and Russell,2001.

Isolation of the At_ALKO4245_cel6A, Ma_ALKO4237_cel6A, Ct_ALKO4265_cel6Aand Te_RF8069_cel6A gene encoding the At_ALKO4245_Cel6A,Ma_ALKO4237_Cel6A, Ct_ALKO4265_Cel6A and Te_RF8069_Cel6A polypeptides isdescribed in Example 1. Briefly, the 1032 bp PCR fragment (SEQ ID NO: 7)and the 831 bp PCR fragment (SEQ ID NO:8) obtained by using thesequences of the degenerate oligonucleotide primers (SEQ ID NO:1 and SEQID NO:2) were used to isolate the At_ALKO4245_cel6A gene from Acremoniumthermophilum ALKO4245 and the Ma_ALKO4237_cel6A gene from Melanocarpusalbomyces ALKO4237 in pCR®4Blunt-TOPO® vector. The full-length A.thermophilum cel6A gene was included in the plasmid pALK2582 depositedin E. coli to the DSMZ culture collection under accession number DSM22946. The full-length M. albomyces cel6A gene was included in theplasmid pALK2581 deposited in E. coli to the DSMZ culture collectionunder accession number DSM 22945.

The Ct_ALKO4265_cel6A gene was isolated using the primer pairs of SEQ IDNO:3 and SEQ ID NO:4 and the Te_RF8069_cel6A gene was isolated using theprimer pairs of SEQ ID NO:5 and SEQ ID NO:6 as described in Example 1.The PCR fragments containing the full-length cel6A genes from Chaetomiumthermophilum ALKO4265 and Talaromyces emersonii RF8069 were included inplasmids pALK2904 and pALK3006 deposited in E. coli to the DSMZ culturecollection under accession numbers DSM 22947 and DSM 23185,respectively.

The deduced amino acid sequences of the CBHII/Cel6A cellobiohydrolaseswere analyzed from the DNA sequences as described in Example 1.

The length of the At_ALKO4245_cel6A (SEQ ID NO: 11) gene, encoding theAcremonium thermophilum CBHII/Cel6A cellobiohydrolase (SEQ ID NO: 12),is 1830 by (including the stop codon). Four putative introns were foundhaving the length of 79, 72, 117 and 126 bps. Thus, the coding region ofAt_ALKO4245_cel6A is 1434 by (stop codon not included) and the deducedprotein sequence consists of 478 amino acids including a predictedsignal sequence of 18 amino acids (SignalP V3.0; Nielsen et al., 1997;Nielsen and Krogh, 1998 and Bendtsen et al., 2004). The deduced aminoacid sequence had homology to the published CBHII/Cel6A sequences whenanalyzed using the BLAST program, version 2.2.9 at NCBI, National Centerfor Biotechnology Information; Altschul et al., 1990). The predictedmolecular mass of the mature enzyme excluding the signal sequence was 48918 Da and the predicted pI was 4.82. These predictions were made usingthe Compute pI/MW tool at ExPASy server (Gasteiger et al., 2003). Theidentity values of the full-length At_ALKO4245_Cel6A sequence to thecorresponding regions of homologous sequences were obtained by using aClone Manager program (version 9) including the functions “Compare twoSequences/Global/Compare sequences as amino acids/BLOSUM62 scoringmatrix”. The values (%) showing identity with the other CBHII/Cel6Acellobiohydrolases of the present invention is shown in Table 6.Identity with the published CBHII/Cel6A amino acid sequences ispresented in Table 7 and Table 8.

The At_ALKO4245_Cel6A of the invention showed highest homology to thefull-length polypeptides of Thielavia terrestris NRRL 8126 (SEQ ID NO: 2in U.S. Pat. No. 7,220,565, Novozymes Inc., US and SEQ ID NO: 49 inWO2009085868, Novozymes A/S, DK), the unnamed protein product ofPodospora anserina DSM 980 (EMBL accession no. XP_(—)001903170), and thededuced endoglucanase 2 precursor of Neurospora crassa OR74A(XM_(—)955677). The identity with the T. terrestris protein was withinthe full-length polypeptide 75%. Identities with P. anserina and N.crassa polypeptides were 69%.

The length of the Ma_ALKO4237_cel6A (SEQ ID NO: 13) gene, encoding theMelanocarpus albomyces CBHII/Cel6A cellobiohydrolase (SEQ ID NO: 14), is1607 bp (including the stop codon). Two putative introns were foundhaving the length of 93 and 95 bps. Thus, the coding region ofMa_ALKO4237_cel6A is 1416 bp (stop codon not included) and the deducedprotein sequence consists of 472 amino acids including a predictedsignal sequence of 17 amino acids (SignalP V3.0; Nielsen et al., 1997;Nielsen and Krogh, 1998 and Bendtsen et al., 2004). The predictedmolecular mass of the mature enzyme excluding the signal sequence was 48627 Da and the predicted pI was 4.50. These predictions were made usingthe Compute pI/MW tool at ExPASy server (Gasteiger et al., 2003). TheMa_ALKO4237_Cel6A of the invention showed highest homology to thefull-length polypeptide of an uncharacterized organism, disclosed as anamino acid sequence SEQ ID NO: 413 in WO2008095033 (Syngenta Inc., US)(72%), to the CBHII polypeptide of Thielavia terrestris NRRL 8126 (SEQID NO:49 in WO2009085868, Novozymes A/S, DK) (71%) and the hypotheticalprotein CHGG_(—)10762 of Chaetomium globosum CBS 148.151 (EMBL accessionno. XP001226029) (75% identity).

The length of the Ct_ALKO4265_cel6A (SEQ ID NO: 9) gene, encoding theChaetomium thermophilum CBHII/Cel6A cellobiohydrolase (SEQ ID NO: 15) is1757 bp (including the stop codon). Three putative introns were foundhaving the length of 77, 196 and 56 bps. Thus, the coding region ofCt_ALKO4265_cel6A is 1425 bp (stop codon not included) and the deducedprotein sequence consists of 475 amino acids including a predictedsignal sequence of 17 amino acids (SignalP V3.0; Nielsen et al., 1997;Nielsen and Krogh, 1998 and Bendtsen et al., 2004). The predictedmolecular mass of the mature enzyme excluding the signal sequence was 49408 Da and the predicted pI was 5.31. The Ct_ALKO4265_Cel6A of theinvention showed highest homology to the deduced cellobiohydrolase offamily 6 of Chaetomium thermophilum CT2 (EMBL accession no. AY861348; CN1757709, Shandong Agricultural University, CN), to the polypeptide of C.thermophilum CGMCC0859 having cellobiohydrolase II activity (SEQ ID NO:2 in EP1578964B1, Novozymes A/S, DK) and to the Chaetomium thermophilumCBHII of SEQ ID NO: 36 (the amino acid sequence of Sequence listing) orSEQ ID NO:46 (the amino acid sequence of description) or SEQ ID NO:45(the nucleotide sequence of description) in WO2009059234, NovozymesInc., US. The identities within the full-length polypeptides were 94%.

The length of the Te_RF8069_cel6A (SEQ ID NO: 10) gene, encoding theTalaromyces emersonii CBHII/Cel6A cellobiohydrolase (SEQ ID NO: 16), is1754 bp (including the stop codon). Seven putative introns were foundhaving the length of 50, 44, 52, 56, 53, 59 and 60 bps. Thus, the codingregion of Te_RF8069_cel6A is 1377 bp (stop codon not included) and thededuced protein sequence consists of 459 amino acids including apredicted signal sequence of 19 amino acids (SignalP V3.0; Nielsen etal., 1997; Nielsen and Krogh, 1998 and Bendtsen et al., 2004). Thepredicted molecular mass of the mature enzyme excluding the signalsequence was 46 618 Da and the predicted pI was 4.27. TheTe_RF8069_Cel6A of the invention showed highest homology to thepolypeptide of Talaromyces emersonii (Q8N1B5 in FIG. 3A-C ofWO2006074005, Novozymes Inc., US) and the cellobiohydrolase II ofTalaromyces emersonii (AY075018). The identities within the full-lengthamino acid sequences were 90%.

Thus, within the scope of the invention is an isolated polynucleotidesequence or isolated nucleic acid molecule, which encodes a CBHII/Cel6Acellobiohydrolase enzyme or polypeptide comprising the amino acidsequence of the full-length form of the At_ALKO4245_Cel6A enzymecharacterized in SEQ ID NO: 12, Ma_ALKO4237_Cel6A enzyme characterizedin SEQ ID NO:14, Ct_ALKO4265 characterized in SEQ ID NO:15 orTe_RF8069_Cel6A characterized in SEQ ID NO:16.

Further, within the scope of the present invention are nucleic acidmolecules or polynucleotide sequences which encode a CBHII/Cel6Apolypeptide having cellobiohydrolase activity and at least 76%,preferably at least 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96% or97%, more preferably at least 98%, most preferably 99% identity to thefull-length amino acid sequence of SEQ ID NO: 12. The identities of thetwo enzymes are compared within the corresponding sequence regions, i.e.within the full-length region of the CBHII/Cel6A polypeptide.

Another preferred embodiment of the invention is a fungal CBHII/Cel6Acellobiohydrolase enzyme showing at least 76%, preferably at least 78%,80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96% or 97%, more preferably atleast 98%, most preferably 99% identity to the full-length amino acidsequence of SEQ ID NO:14.

Further, preferred CBHII/Cel6A cellobiohydrolases show at least 95%,preferably at least 96% or least 97%, even more preferably at least 98%identity, most preferably at least 99% identity to the full-length aminoacid sequence of SEQ ID NO: 15.

Other preferred CBHII/Cel6a polypeptides show at least 91%, preferablyat least 92%, 93%, 94%, 95%, 96% or 97%, more preferably at least 98%,and most preferably 99% identity to the full-length amino acid sequenceof SEQ ID NO: 16.

Within the scope of the invention is an isolated nucleic acid moleculewhich comprises a polynucleotide sequence encoding a polypeptide, whichhas the amino acid sequence of the full-length CBHII/Cel6Acellobiohydrolase of the invention as well as fragments or natural orsynthetic variants of the polypeptides of the invention which havecellobiohydrolase activity and properties similar to the full-lengthpolypeptides. Such fragment may, for example be an enzyme which lacksthe secretion signal sequence or carbohydrate binding domain or CBD.

One preferred embodiment of the invention is an isolated nucleic acidmolecule comprising a “coding sequence” of the polynucleotide sequenceincluded in SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:9 or SEQ ID NO:10 or asubsequence thereof. According to one preferred embodiment of theinvention the polypeptide is encoded by the nucleic acid molecule havingthe nucleotide sequence SEQ ID NO: 11 comprising the coding sequence forthe At_ALKO4245_Cel6A enzyme. The expression “coding sequence” means thenucleotide sequence which initiates from the translation start codon(ATG) and stops at the translation stop codon (TAA, TAG or TGA) and maycomprise intron regions. The translated full-length polypeptide startsusually with methionine.

According to another preferred embodiment of the invention the isolatednucleic acid molecule comprises a coding sequence of a polynucleotidesequence included in plasmid pALK2582 deposited in Escherichia coliRF8175 under accession number DSM 22946, plasmid pALK2581 deposited inEscherichia coli RF8174 under accession number DSM 22945, plasmidpALK2904 deposited in Escherichia coli RF8214 under accession number DSM22947, or plasmid pALK3006 deposited in Escherichia coli RF8333 underaccession number DSM 23185, said polynucleotide sequences encoding theAt_ALKO4245_Cel6A, Ma_ALKO4237_Cel6A, Ct_ALKO4265_Cel6A andTe_RF8069_Cel6A cellobiohydrolases, respectively.

The nucleic acid molecule of the invention may also be an analogue ofthe nucleotide sequence characterized above. The “degeneracy” meansanalogues of the nucleotide sequence, which differ in one or morenucleotides or codons, but which encode the recombinant CBHII/Cel6A ofthe invention.

The nucleic acid molecule may also be a nucleic acid molecule orpolynucleotide sequence hybridizing under stringent conditions to apolynucleotide sequence contained in plasmids pALK2582, pALK2581,pALK2904 or pALK3006 deposited in E. coli under the accession numbersDSM 22946, DSM 22945, DSM 22947 or DSM 23185 or a subsequence thereof,and encoding a polypeptide having cellobiohydrolase activity and anamino acid sequence which within the corresponding sequence region showsat least 76% identity to the full-length amino acid sequence as depictedin SEQ ID NO:12, at least 76% identity to the full-length amino acidsequence of SEQ ID NO:14, at least 95% identity to the full-length aminoacid sequence of SEQ ID NO:15 or at least 91% to the full-length aminoacid sequence of SEQ ID NO:16. The hybridizing DNA may originate from afungus belonging to genus Acremonium, Melanocarpus, Chaetomium orTalaromyces or it may originate from other fungal species.

According to a preferred embodiment of the invention the fungalCBHII/Cel6A cellobiohydrolase enzyme is encoded by an isolated nucleicacid molecule, which comprises the nucleotide sequence of SEQ ID NO:11encoding the full-length form of the At_ALKO4245_Cel6A enzyme of SEQ IDNO:12.

The present invention relates also to a recombinant expression vector orrecombinant expression construct, which can be used to propagate orexpress the nucleic acid molecule or polynucleotide sequence encodingthe chosen CBHII/Cel6A cellobiohydrolase in a suitable prokaryotic oreukaryotic host. The recombinant expression vector comprises DNA ornucleic acid sequences which facilitate or direct expression andsecretion of the CBHII/Cel6A polypeptide encoding sequence or gene in asuitable host, such as promoters, enhancers, terminators (includingtranscription and translation termination signals) and signal sequencesoperably linked the polynucleotide sequence encoding said polypeptide.The expression vector may further comprise marker genes for selection ofthe transformant strains or the selection marker may be introduced tothe host in another vector construct by co-transformation. Saidregulatory sequences may be homologous or heterologous to the productionorganism or they may originate from the organism, from which the geneencoding the CBHII/Cel6A polypeptide is isolated.

Examples of promoters for expressing the CBHII/Cel6A of the invention infilamentous fungal hosts are the promoters of A. oryzae TAKA amylase,alkaline protease ALP and triose phosphate isomerase, Rhizopus mieheilipase, Aspergillus niger or A. awamori glucoamylase (glaA), Fusariumoxysporum trypsin-like protease, Chrysosporium lucknowensecellobiohydrolase 1 promoter, Trichoderma reesei cellobiohydrolase I(Cel7A) etc.

In yeast, for example promoters of S. cerevisiae enolase (ENO-1),galactokinase (GAL1), alcohol dehydrogenase (ADH2) and3-phosphoglycerate kinase can be used to provide expression.

Examples of promoter sequences for directing the transcription of theCBHII/Cel6A polypeptide of the invention in a bacterial host are thepromoter of lac operon of Escherichia coli, the Streptomyces coelicoloragarase dagA promoter, the promoter of the B. licheniformisalpha-amylase gene (amyL), the promoter of the B. stearothermophilusmaltogenic amylase gene (amyM), the promoters of the B. sublitis xylAand xylB genes, etc.

Suitable terminators include those of the above mentioned genes or anyother characterized terminator sequences.

Suitable transformation or selection markers include those whichcomplement a defect in or adds a new feature, e.g. enzyme activity tothe host, for example the dal genes from B. subtilis or B. licheniformisor Aspergillus amdS and niaD. The selection may be based also on a tomarker conferring antibiotic resistance, such as ampicillin, kanamycin,chloramphenicol, tetracycline, phleomycin or hygromycin resistance.

Extracellular production of the CBHII/Cel6A of the invention ispreferable. Thus, the recombinant vector comprises sequencesfacilitating secretion in the selected host. The signal sequence of theCBHII/Cel6A cellobiohydrolase of the invention or the presequence orprepeptide may be included in the recombinant expression vector or thenatural signal sequence may be replaced with another signal sequencecapable of facilitating expression in the selected host. Thus, thechosen signal sequence may be homologous or heterologous to theexpression host.

Examples of suitable signal sequences are those of the fungal or yeastorganisms, e.g. signal sequences from well-expressed genes. Such signalsequences are well known from the literature.

The recombinant vector may further comprise sequences facilitatingintegration of the vector into the host chromosomal DNA to obtain stableexpression. The vector may also be a fusion construct and comprisesequences encoding a carrier polypeptide, which is genetically fusedwith the same coding sequence as the coding sequence of the protein ofinterest and which improves secretion of the polypeptide in aheterologous host organism or which facilitates the protein purificationafter production. Such carriers include, e.g. the proteins produced inhigh amounts by the native host, such as the cellulases of T. reesei orglucoamylases of Aspergillus species. The carrier polypeptide may alsobe an intact domain of the secretable protein, such as the CBD. Thecarrier protein and the protein of interest may remain as a fusion afterthe secretion, or are separated by a proteolytic processing during theprotein export process in the host, or are separated chemically orbiochemically after the secretion into the supernatant.

The At_ALKO4245_Cel6A, Ma_ALKO4237_Cel6A, Ct_ALKO4265_Cel6A andTe_RF8069_Cel6A cellobiohydrolase of the invention were expressed withtheir own signal sequence from the Trichoderma reesei cbh1 (cel7A)promoter as described in Example 2. The expression construct used totransform the T. reesei host included also cbh1 terminator and amdSmarker for selecting transformants from untransformed cells.

The present invention relates also to host cells comprising therecombinant expression vector as described above. Suitable hosts forproduction of the fungal CBHII/Cel6A cellobiohydrolase are homologous orheterologous hosts, such as the microbial hosts including bacteria,yeasts and fungi. Production systems in plant or mammalian cells arealso possible.

Filamentous fungi, such Trichoderma, Aspergillus, Fusarium, Humicola,Chrysosporium, Neurospora, Rhizopus, Penicillium and Mortiriella, arepreferred production hosts due to the ease of down-stream processing andrecovery of the enzyme product. Suitable expression and production hostsystems are for example the production system developed for thefilamentous fungus host Trichoderma reesei (EP 244234), or Aspergillusproduction systems, such as A. oryzae or A. niger (WO 9708325, U.S. Pat.No. 5,843,745, U.S. Pat. No. 5,770,418), A. awamori, A. sojae and A.japonicus-type strains, or the production system developed for Fusarium,such as F. oxysporum (Malardier et al., 1989) or F. venenatum, and forNeurospora crassa, Rhizopus miehei, Mortiriella alpinis, H. lanuginosaor H. insolens or for Chrysosporium lucknowense (U.S. Pat. No.6,573,086). Suitable production systems developed for yeasts are systemsdeveloped for Saccharomyces, Schizosaccharomyces or Pichia pastoris.Suitable production systems developed for bacteria are a productionsystem developed for Bacillus, for example for B. subtilis, B.licheniformis, B. amyloliquefaciens, for E. coli, or for theactinomycete Streptomyces. Preferably the CBHII/Cel6A cellobiohydrolaseof the invention is produced in a filamentous fungal host of the genusTrichoderma or Aspergillus, such as T. reesei, or A. niger, A. oryzae,A. sojae, A. awamori or A. japonicus-type strains. According the mostpreferred embodiment of the invention the fungal CBHII/Cel6Acellobiohydrolase is produced in T. reesei.

The production host cell may be homologous or heterologous to theCBHII/Cel6A cellobiohydrolase of the invention. Preferably therecombinant host is modified to express and secrete cellulolytic enzymesas its main activity or one of its main activities. This can be done bydeleting major homologous secreted genes e.g. the four major cellulasesof Trichoderma and by targeting heterologous genes to a locus that hasbeen modified to ensure high expression and production levels. Forexample, the host may be free of homogenous cellobiohydrolases due toremoval of said cellobiohydrolases either by inactivation or removal ofone or more host cellobiohydrolases, e.g. by deletion of the gene(s)encoding such homogenous or homologous cellobiohydrolase(s).

The present invention relates also to a process for producing aCBHII/Cel6A polypeptide having cellobiohydrolase activity, said processcomprising the steps of culturing the natural or recombinant host cellcarrying the recombinant expression vector for a CBHII/Cel6Acellobiohydrolase of the invention under suitable conditions andoptionally isolating said enzyme. The production medium may be a mediumsuitable for growing the host organism and containing inducers forefficient expression. Suitable media are well-known from the literature.

The invention relates to a polypeptide having cellobiohydrolaseactivity, said polypeptide being encoded by the nucleic acid molecule ofthe invention and which is obtainable by the process described above.

The invention further relates to a process for obtaining an enzymepreparation comprising a CBHII/Cel6A polypeptide, which hascellobiohydrolase activity, said process comprising the steps ofculturing a host cell carrying the expression vector of the inventionand preparing the whole culture broth, or separating the cells from thespent culture medium and obtaining the supernatant havingcellobiohydrolase activity.

The present invention relates also to an enzyme preparation, whichcomprises the CBHII/Cel6A enzyme characterized above. The enzymepreparation or composition has cellobiohydrolase activity and isobtainable by the process according to the invention.

Within the invention is an enzyme preparation which comprises the fungalCBHII/Cel6A cellobiohydrolase of the invention.

Said enzyme preparation may further comprise different types of enzymesin addition to the CBHII/Cel6A cellobiohydrolase of this invention, forexample another cellulase including a cellobiohydrolase, endoglucanaseand beta-glucosidase, beta-glucanase, an amylase, a lipase, cutinase, aprotease, xylanase, beta-xylosidase, mannanase, beta-mannosidase,α-glucuronidase, acetyl xylan esterase, α-arabinofuranosidase,α-galactosidase, pectinase, involving endo- and exo-α-L-arabinases,α-galactosidase, endo- and exo-galactoronase, xyloglucanase,endopectinlyase, pectate lyase, and pectinesterase, phenol esterase,ligninase involving lignin peroxidase, manganese-dependent peroxidase,H₂O₂-generating enzyme and/or an oxidase such as a laccase or peroxidasewith or without a mediator. These enzymes are expected to enhance theperformance of the CBHII/Cel6A enzyme of the invention by removing thecarbohydrates, proteins and oils or fats present in the material to behandled. Said enzymes may be natural or recombinant enzymes produced bythe host strain or may be added to the culture supernatant after theproduction process. The enzyme compositions or enzyme preparations maycontain any combination of these enzymes. The CBHII/Cel6Acellobiohydrolases may also be used in combination to commerciallyavailable enzyme preparations.

The enzymes needed for the hydrolysis of the cellulosic materialaccording to the invention may be added in an enzymatically effectiveamount either simultaneously, e.g. in the form of an enzyme mixture, orsequentially, or as a part of the simultaneous saccharification andfermentation (SSF) process or they may be produced by the fermentativemicroorganism in the consolidated bioprocess.

The enzyme preparation or composition may contain the enzymes in atleast partially purified and isolated form. It may even essentiallyconsist of the desired enzyme or enzymes.

Alternatively the preparation may be a whole culture broth or spentculture medium or filtrate containing one or more desired enzymes. In aconsolidated bioprocess the enzymes may be produced by the fermentativemicroorganism used in the process. In addition to the enzymaticactivity, the preparation may contain additives, such as mediators,stabilizers, buffers, preservatives, surfactants and/or culture mediumcomponents.

Preferred additives are such, which are commonly used in enzymepreparations intended for a particular application. Surfactants areuseful in emulsifying grease and wetting surfaces. The surfactant may bea non-ionic including semi-polar and/or anionic and/or cationic and/orzwitterionic. Buffers may be added to the enzyme preparation to modifypH or affect performance or stability of other ingredients. Suitablestabilizers include polyols such as propylene glycol or glycerol, asugar or sugar alcohol, lactic acid, boric acid, or boric acidderivatives, peptides, etc. Bleaching agent is used to oxidize anddegrade organic compounds. Examples of suitable chemical bleachingsystems are H₂O₂ sources, such as perborate or percarbonate with orwithout peracid-forming bleach activators such astetra-acetylethylenediamine, or alternatively peroxyacids, e.g. amide,imide or sulfone type. Chemical oxidizers may be replaced partially orcompletely by using oxidizing enzymes, such as laccases or peroxidases.Many laccases do not function effectively in the absence of mediators.Builders or complexing agents include substances, such as zeolite,diphosphate, triphosphate, carbonate, citrate, etc. The enzymepreparation may further comprise one or more polymers, such ascarboxymethylcellulose, poly(ethylene glycol), poly(vinyl alcohol),poly(vinylpyrrolidone), etc. Also, softeners, caustics, preservativesfor preventing spoilage of other ingredients, abrasives and substancesmodifying the foaming and viscosity properties can be added.

According to one preferred embodiment of the invention said enzymepreparation is in the form of spent culture medium, liquid, powder orgranulate. Preferably the enzyme preparation is spent culture medium.

The present invention relates also to various uses of the fungalCBHII/Cel6A cellobiohydrolase of the invention, in which hydrolysis ormodification of cellulosic material is desired. Such uses include anyapplication, in which cellulolytic enzymes are conventionally used, suchas in fuel, textile, detergent, pulp and paper, food, feed or beverageindustry. Addition of the fungal CBHII/Cel6A cellobiohydrolase of theinvention to an enzyme composition comprising other cellulose degradingenzymes such as cellobiohydrolase I, endoglucanases andbeta-glucosidases, greatly enhances the hydrolysis and leads to almosttotal hydrolysis of the polymeric cellulose backbone to glucosemonomers. The major product of CBHII/Cel6A action is cellobiose composedof two glucose units.

One preferred embodiment is the use of the method of the invention inapplications requiring stability/performance of the CBHII/Cel6Acellobiohydrolase at moderate/conventional or to elevated temperatures,i.e. requiring thermophilic or thermostable enzymes. Elevatedtemperatures are known to enhance the hydrolysis of crystallinecellulose present in cellulosic or lignocellulosic materials, thusreducing the total amount of enzymes needed in hydrolysis or reducingthe required hydrolysis time. Also, since at elevated temperatures theviscosity of the lignocellulosic substrate is decreased, thermostableenzymes make it possible to work at higher solid loadings and save ininvestment costs.

Another preferred embodiment is the use of the CBHII/Cel6A polypeptideof the invention or the enzyme preparation comprising said polypeptidein hydrolyzing cellulosic material for the production of biofuelcomprising ethanol, propanol, butanol and alike. In production ofbiofuel the CBHII/Cel6A cellobiohydrolases of the invention are likeother cellulolytic enzymes especially suitable for producing glucosemonomers from the polymeric cellulosic material which may then befermented by yeast strains into ethanol, and used as fuel.

The lignocellulosic material may be pretreated before the enzymatichydrolysis to disrupt the fiber structure of cellulosic substrates andmake the cellulose fraction more accessible to the cellulolytic enzymes.Current pretreatments include mechanical, chemical or thermal processesand combinations thereof. The material may for example be pretreated bysteam explosion or acid hydrolysis. The saccharification process, i.e.production of sugar monomers, and fermentation by yeast strains may beperformed separately or simultaneously in the same reactor. TheCBHII/Cel6A cellobiohydrolase of the invention has a great advantage tothe commercial enzymes presently for sale in that it is stable also atelevated temperatures, i.e. is thermostable. Hydrolysis of cellulosic orlignocellulosic materials is known to enhance at elevated temperaturesand yield sugar monomers more efficiently.

The use of the CBHII/Cel6A polypeptides of the invention enables the useof high biomass consistency and lead to high sugar and ethanolconcentrations. This approach may lead to significant savings in energyand investments costs. The high temperature also decreases the risk ofcontamination during hydrolysis.

The sugar hydrolysates may also serve as raw material for othernon-microbial processes, e.g., for enrichment, isolation andpurification of high value sugars or various polymerization processes.

The glucose monomers can also be used as intermediates or raw materialsfor the production of various chemicals or building blocks for theprocesses of chemical industry, e.g. in so called biorefinery.

In the pulp and paper industry the polypeptides may be used to modifycellulosic fiber for example in treating kraft pulp, mechanical pulp, orrecycled paper.

In textile industry the CBHII/Cel6A cellobiohydrolases find applicationsin softening and/or improving the feel of cotton fabrics and removingindigo dyes in replacement of stone washing.

In feed industry the CBHII/Cel6A cellobiohydrolases of the invention maybe used in degrading the cellulosic or hemicellulosic materials presentin feedstocks.

In detergent industry the CBHII/Cel6A cellobiohydrolase may be used inhand or machine laundry or dishwashing compositions for removal ofcellulosic stains.

The invention is illustrated by the following non-limiting examples.From the experimental results it can be concluded that the fungalCBHII/Cel6A cellobiohydrolases of the invention are capable ofsatisfying the greatly varying demands of different industry requiringefficient hydrolysis of cellulosic material present in varyingfeedstocks.

Example 1 Cloning of the Cellobiohydrolase 2 (cbh2) Genes

Standard molecular biology methods were used in the isolation and enzymetreatments of DNA (e.g. isolation of plasmid DNA, digestion of DNA toproduce DNA fragments), in E. coli transformations, sequencing etc. Thebasic methods used were either as described by the enzyme, reagent orkit manufacturer or as described in the standard molecular biologyhandbooks, e.g. Sambrook and Russell (2001). Isolation of genomic DNAwas performed as described in detail by Raeder and Broda (1985).

Four thermophilic fungal strains from the Roal Oy culture collectionwere selected for cloning based on the previous findings that thestrains produce thermostable cellulases (WO2007071818; Maheshwari etal., 2000; Murray et al., 2003; Miettinen-Oinonen et al., 2004). Theprobes for cloning the cbh2 genes from Acremonium thermophilum ALKO4245and Melanocarpus albomyces ALKO4237 were synthesised by PCR. Degenerateoligos were planned basing on the alignment of the previously publishedamino acid sequences of cellobiohydrolase II (CBHII) proteins. Thesequences of the homologous primers for the cloning of the cbh2 genesfrom Chaetomium thermophilum ALK4265 and Talaromyces emersonii RF8069strains were obtained from the published nucleotide sequences (AY861348;DQ020255; CQ838150; Murray et al., 2003; AY075018; AF439936). Thesequences of the primers are shown in Table 2 (SEQ ID NOs: 1-6).

TABLE 2  The oligonucleotides used as PCR primers to amplifyprobes for screening of cbh2 genes from Acremoniumthermophilum ALKO4245 and Melanocarpus albomyces ALKO4237 and to amplify full-length cbh2 genes from Chaetomiumthermophilum ALKO4265 and Talaromyces emersonii RF8069. Template,genomic Oligonu- Length DNA from cleotide (bp) Sequence^((a) SEQ ID NO:ALKO4245 CBH_1S 17 TGGGGNCARTGYGGNGG (s) 1 CBH_1AS 17GCNGGCCANCCNARCCA (as) 2 ALKO4237 CBH_1S 17 TGGGGNCARTGYGGNGG (s) 1CBH_1AS 17 GCNGGCCANCCNARCCA (as) 2 ALKO4265 CBH_8 21ATGGCTAAGCAGCTGCTGCTC (s) 3 CBH_9 20 TCAGARCGGAGGGTTGGCAT (as) 4 RF8069Te_CBH_A 44 TATTATCCGCGGACTGCGCATCA 5 TGCGGAATCTTCTTGCTCTTG (s) Te_CBH_B38 AATTTGGATCCTCAGAACAGCG 6 GGTTAGCATTCGTGAG (as) ^((a)R = A or G, N = Aor G or T or C, Y = T or C; “s” in the parenthesis = sense strand, “as”in the parenthesis = antisense strand.

The probes were amplified by PCR with primers described in Table 2 usingthe genomic DNA as a template in the reactions. The PCR mixtures ofAcremonium thermophilum ALKO4245 and Melanocarpus albomyces ALKO4237contained 10 mM Tris-HCl, pH 8.8, 50 mM KCl, 0.1% Triton X-100, 1.5 mMMgCl₂, 0.2 mM dNTPs, 5 μM each primer and 1-2 units of Dynazyme EXT DNApolymerase (Finnzymes, Finland) and 0.5-1 μg of the correspondinggenomic DNA. The conditions for the PCR reactions were the following: 5min initial denaturation at 95° C., followed by 30 cycles of 1 min at95° C., 1 min annealing at 60° C. (±5° C. gradient), 2 min extension at72° C. and a final extension at 72° C. for 10 min. For PCR-cloning ofthe cbh2 genes from Chaetomium thermophilum ALKO4265 and Talaromycesemersonii RF8069 strains, the reactions contained 1× Phusion GC and 1×Phusion HF buffers, respectively, with 0.2 mM dNTPs, 5 μM each primerand 1-2 units of Phusion DNA polymerase (Finnzymes, Finland) and 0.5-1μg of the corresponding genomic DNA. The conditions for the PCRreactions were the following: 3 min initial denaturation at 98° C.,followed by 30 cycles of 30 sec at 98° C., 30 sec annealing at 55° C.(±5° C. gradient), 1-2 min extension at 72° C. and a final extension at72° C. for 10 min.

Primer combinations described in Table 2 produced a specific DNA producthaving the expected size (according to calculations basing on publishedcbh2 sequences). The DNA products were isolated and purified from thePCR reaction mixtures and cloned to pCe4Blunt-TOPO® vector according tothe manufacturer's instructions (Invitrogen, USA). The inserts werecharacterized by sequencing and by performing Southern blothybridizations to the genomic DNAs digested with several restrictionenzymes. The PCR fragments, which were chosen to be used as probes forgene cloning from the Acremonium thermophilum ALKO4245 and Melanocarpusalbomyces ALKO4237 strains are presented in Table 3. In addition, thetable describes the PCR fragments containing the full-length cbh2 genesfrom Chaetomium thermophilum ALKO4265 and Talaromyces emersonii RF8069strains.

TABLE 3 The primers used in the PCR reactions, probes chosen forscreening of the cbh2 genes from Acremonium thermophilum ALKO4245 andMelanocarpus albomyces ALKO4237 and DNA fragments containing thefull-length cbh2 genes from Chaetomium thermophilum ALKO4265 andTalaromyces emersonii RF8069. The genomic template DNA and the name ofthe plasmid containing the probe fragment are shown. Genomic DNA used asa template Fragment SEQ Forward Reverse in PCR obtained Insert in IDGene primer primer reaction (kb) plasmid NO: ALKO4245_cel6A CBH_1SCBH_1AS ALKO4245 1.0 kb pALK2580 7 ALKO4237_cel6A CBH_1S CBH_1ASALKO4237 0.8 kb pALK2576 8 ALKO4265_cel6A CBH_8 CBH_9 ALKO4265 1.8 kbpALK2904 9 RF8069_cel6A Te_CBH_A Te_CBH_B RF8069 1.8 kb pALK3006 10

The deduced amino acid sequences from all these PCR fragments hadhomology to the published CBHII/Cel6A sequences (BLAST program, version2.2.9 at NCBI, National Center for Biotechnology Information; Altschulet al., 1990). The 1757 bp PCR fragment in pALK2904 plasmid (SEQ ID NO:9) and the 1788 bp PCR fragment in pALK3006 plasmid (SEQ ID NO: 10)contained the full-length cbh2/cel6A genes from Chaetomium thermophilumALKO4265 and Talaromyces emersonii RF8069, respectively. The geneencoding the Chaetomium thermophilum ALKO4265 was named asCt_ALKO4265_cel6A and Talaromyces emersonii RF8069 gene was named asTe_RF8069_cel6A. The E. coli strains RF8214 (=pALK2904) and RF8333(=pALK3006) were deposited to the DSM collection under the accessionnumbers DSM 22947 and DSM 23185, respectively.

Acremonium thermohilum ALKO4245 and Melanocarpus albomyces ALKO4237genomic DNAs were digested with several restriction enzymes for Southernblot analysis. The probes for the hybridizations were the 1032 bp (SEQID NO: 7) and 831 bp (SEQ ID NO:8) EcoRI fragments, cut from theplasmids pALK2580 and pALK2576, respectively. The above probes werelabeled by using digoxigenin according to supplier's instructions(Roche, Germany). Hybridizations were performed over night at 68° C.After hybridization the filters were washed 2×5 min at RT using2×SSC-0.1% SDS followed by 2×15 min at 68° C. using 0.1×SSC-0.1% SDS.

From the genomic DNA of Acremonium thermophilum ALKO4245, approximate 8kb XbaI-digested fragment was hybridized using dioxigenin-labeled 1032bp EcoRI fragment from the pALK2580 as a probe. Correspondingly, about4.5 kb BamHI-digested fragment was hybridized with dioxigenin-labeled831 bp EcoRI fragment of the pALK2576 from the genomic DNA of theMelanocarpus albomyces ALKO4237. The hybridizing genomic DNA fragmentswere isolated from the pool of the digested genomic fragments based ontheir size. The genomic fragments were isolated from agarose gel andwere cloned to pBluescript II KS+ (Stratagene, USA) vectors cleaved witheither XbaI (Acremonium thermophilum ALKO4245) or BamHI (Melanocarpusalbomyces ALKO4237). Ligation mixtures were transformed to Escherichiacoli XL10-Gold cells (Stratagene) and plated on LB (Luria-Bertani)plates containing 50-100 μg/ml ampicillin. The E. coli colonies werescreened for positive clones using colonial hybridization with thepALK2580 and pALK2576 inserts as probes in the hybridization conditionscorrespondingly to that described above for Southern blot analyses,except using the hybridization temperature of 65° C. instead of 68° C.Several positive clones were collected from the plates. They were shownby restriction digestion to contain inserts of expected sizes. Thefull-length gene encoding the Acremonium thermohilum ALKO4245CBHII/Cel6A (At_ALKO4245_Cel6A, SEQ ID NO: 11) was sequenced from the 7kb XbaI insert and the plasmid containing this insert was namedpALK2582. The E. coli strain RF8175 including the plasmid pALK2582 wasdeposited to the DSM collection under the accession number DSM 22946.The gene encoding the Acremonium thermohilum ALKO4245 protein was namedas At_ALKO4245_cel6A. Correspondingly, the full-length gene encoding theMelanocarpus albomyces ALKO4237 CBHII/Cel6A (Ma_ALKO4237_Cel6A, SEQ IDNO: 12) was sequenced from the 5 kb BamHI insert and the plasmidcontaining this insert was named pALK2581. The E. coli strain RF8174including the plasmid pALK2581 was deposited to the DSM collection underthe accession number DSM 22945. The gene encoding the Melanocarpusalbomyces ALKO4237 was named as Ma_ALKO4237_cel6A. The relevantinformation on the genes and the deduced protein sequences (SEQ ID NOs:9-16) are summarized in Table 4 and Table 5, respectively.

TABLE 4 The summary on the cbh2/cel6A genes isolated from Acremoniumthermophilum ALKO4245, Melanocarpus albomyces ALKO4237, Chaetomiumthermophilum ALKO4265 and Talaromyces emersonii RF8069. Length withCoding No of Lengths of introns region putative putative introns SEQ IDGene (bp) ^((a) (bp) ^((b) introns (bp) NO: At_ALKO4245_cel6A 1830 14344 79, 72, 117, 126 11 Ma_ALKO4237_cel6A 1607 1416 2 93, 95 13Ct_ALKO4265_cel6A 1757 1425 3 77, 196, 56 9 Te_RF8069_cel6A 1754 1377 750, 44, 52, 56, 10 53, 59, 60 ^((a) The STOP codon is included. ^((b)The STOP codon is not included.

TABLE 5 The summary of the amino acid sequences deduced from thecbh2/cel6A genes sequences from Acremonium thermophilum ALKO4245,Melanocarpus albomyces ALKO4237, Chaetomium thermophilum ALKO4265 andTalaromyces emersonii RF8069. Predicted No Length MW PredictedCBHII/Cel6A of of ss (Da), ss pI, ss not SEQ ID protein aas NN/HMM^((a)CBD^((b) not incl^((c) incl NO: At_ALKO4245_Cel6A 478 18 Q26 to 489184.82 12 L63 Ma_ALKO4237_Cel6A 472 17 Q25 to 48627 4.50 14 L62Ct_ALKO4265_Cel6A 475 17 Q25 to I62 49408 5.31 15 Te_RF8069_Cel6A 459 19Q20 to 46618 4.27 16 V55 ^((a)The prediction on the signal sequence wasmade using the program SignalP V3.0 (Nielsen et al., 1997; Nielsen andKrogh, 1998; Bendtsen et al., 2004); the NN value was obtained usingneural networks. ^((b)The cellulose-binding domain (CBD), the aminoacids of the CBD region are indicated [M1(Met #1) included innumbering]. ^((c)The predicted signal sequence was not included. Theprediction was made using the Compute pI/MW tool at ExPASy server(Gasteiger et al., 2003).

The comparison of the deduced CBHII/Cel6A sequences from Acremoniumthermophilum ALKO4245, Melanocarpus albomyces ALKO4237, Chaetomiumthermophilum ALKO4265 and Talaromyces emersonii DSM 2432 (RF8069) toeach other is presented in Table 6. A program of Clone Manager (version9) including the functions “Compare Two Sequences/Global/Comparesequences as amino acids/BLOSUM62 scoring matrix” was used fordetermining the degree of identity.

TABLE 6 The identity values (%) obtained from alignment of the deducedCBHII/Cel6A amino acid sequences from Acremonium thermophilum ALKO4245,Melanocarpus albomyces ALKO4237, Chaetomium thermophilum ALKO4265 andTalaromyces emersonii RF8069. ALKO4245_Cel6A ALKO4237_Cel6AALKO4265_Cel6A RF8069_Cel6A ALKO4245_Cel6A 100 67 68 66 ALKO4237_Cel6A100 71 60 ALKO4265_Cel6A 100 61 RF8069_Cel6A 100 The full-length aminoacid sequences including the signal sequences were aligned. A program ofClone Manager 9 (Compare Two Sequences/Global/Compare sequences as aminoacids/BLOSUM62 scoring matrix) was used for determining the degree ofidentity.

The comparison of the deduced CBHII/Cel6A sequences from Acremoniumthermophilum ALKO4245, Melanocarpus albomyces ALKO4237, Chaetomiumthermophilum ALKO4265 and Talaromyces emersonii RF8069 to the sequencesfound from the databases are shown in Tables 7 and 8.

TABLE 7 The highest identity sequences to the deduced CBHII/Cel6A aminoacid sequences from Acremonium thermophilum ALKO4245, Melanocarpusalbomyces ALKO4237, Chaetomium thermophilum ALKO4265 and Talaromycesemersonii RF8069. Organism and accession number Identity (%)At_ALKO4245_Cel6A 100 Podospora anserina, XP_001903170 69 Neurosporacrassa, XM_955677 69 Ma_ALKO4237_Cel6A 100 Chaetomium globosum,XP_001226029 75 Ct_ALKO4265_Cel6A 100 Chaetomium thermophilum, AY86134894 Te_RF8069_Cel6A 100 Talaromyces emersonii, AY075018 90 Thefull-length amino acid sequences including the signal sequences werealigned. The database search was performed using BLAST (tblastn, nr/ntdatabase), and Clone Manager 9 program (Compare TwoSequences/Global/Compare sequences as amino acids/BLOSUM62 scoringmatrix) was used for determining the degree of identity.

TABLE 8 The highest identity patent sequences to the deduced CBHII/Cel6Aamino acid sequences from Acremonium thermophilum ALKO4245, Melanocarpusalbomyces ALKO4237, Chaetomium thermophilum ALKO4265 and Talaromycesemersonii RF8069. Organism and accession number Identity (%)At_ALKO4245_Cel6A 100 U.S. Pat. No. 7,220,565 B2, SEQ ID: 2 75WO2009085868 A1, SEQ ID: 49 75 Ma_ALKO4237_Cel6A 100 WO2008095033 A2,SEQ ID: 413 72 WO2009085868 A1, SEQ ID: 49 71 Ct_ALKO4265_Cel6A 100EP1578964 B1, SEQ ID: 2 94 WO2009059234 A2, SEQ ID: 45*⁾ 94 CN1757709A94 Te_RF8069_Cel6A 100 W02006074005 A2, FIG. 3A-C 90 The full-lengthamino acid sequences including the signal sequences were aligned. TheChemical Abstracts Service (CAS) Registry System and Patented ProteinSequences NCBI database searches were performed using BLAST, and CloneManager 9 program (Compare Two Sequences/Global/Compare sequences asamino acids/BLOSUM62 scoring matrix) was used for determining the degreeof identity. *⁾WO200959234 description refers to SEQ ID NO: 45 (DNA) orSEQ ID NO: 46 (protein). WO200959234 Sequence listing refers to SEQ IDNO: 35 (DNA) or SEQ ID NO: 36 (protein).

Example 2 Production of Recombinant CBHII/Cel6A Proteins in Trichodermareesei

Expression plasmids were constructed for production of recombinantCBHII/Cel6A proteins from Acremonium thermophilum ALKO4245, Melanocarpusalbomyces ALKO4237, Chaetomium thermophilum ALKO4265 and Talaromycesemersonii RF8069 in Trichoderma reesei. The expression plasmidsconstructed are listed in Table 9. The recombinant cbh2/cel6A genes,including their own signal sequences, were exactly fused to the T.reesei cbh1/cel7A promoter by PCR. The transcription termination wasensured by the T. reesei cbh1/cel7A terminator and the A. nidulans amdSmarker gene was used for selection of the transformants as described inPaloheimo et al. (2003). The linear expression cassettes (FIG. 1) wereisolated from the vector backbones after EcoRI or EcoRI-SpeI digestionand were transformed into T. reesei protoplasts. The host strain useddoes not produce any of the four major T. reesei cellulases (CBHI,CBHII, EGI, EGII). The transformations were performed as in Penttilä etal. (1987) with the modifications described in Karhunen et al. (1993),selecting acetamide as a sole nitrogen source (amdS marker gene). Thetransformants were purified on selection plates through single conidiaprior to sporulating them on PD.

TABLE 9 The expression cassettes constructed to produce CBHII/Cel6Arecombinant proteins from Acremonium thermophilum ALKO4245, Melanocarpusalbomyces ALKO4237, Chaetomium thermophilum ALKO4265 and Talaromycesemersonii RF8069 in Trichoderma reesei. Cellobiohydrolase II ExpressionExpression protein plasmid cassette ^((a) Terminator ^((b)At_ALKO4245_Cel6A pALK2906 9.4 kb EcoRI 125 bp (XbaI) Ma_ALKO4237_Cel6ApALK2901 9.3 kb EcoRI-SpeI 258 bp (DraIII) Ct_ALKO4265_Cel6A pALK29039.2 kb EcoRI Te_RF8069_Cel6A pALK3010 7.9 kb EcoRI The overall structureof the expression cassettes was as described in FIG. 1. The clonedcbh2/cel6A genes were exactly fused to the T. reesei cbh1/cel7Apromoter. ^((a) The expression cassette for T. reesei transformation wasisolated from the vector backbone by using either EcoRI or EcoRI-SpeIdigestion. ^((b) The number of the nucleotides after the STOP codon ofthe cloned recombinant gene that was included in the expressioncassette. The restriction site at the 3′-end of the genomic genefragment that was used in the construction of the expression cassette isindicated in parenthesis. The Ct_ALKO4265_cel6A gene fragment wasexcised from the 3′-end by EcoRI (a site present in thepCR ®4Blunt-TOPO ® vector). Correspondigly, the Te_RF8069_cel6A genefragment was excised from its 3′-end by BamHI (a site created after stopcodon in PCR). This leaves no original Ct_ALKO4265_cel6A orTe_RF8069_cel6A terminator in the constructs prior to the cbhlterminator sequence.

The CBHII/Cel6A production of the transformants was analyzed from theculture supernatants of the shake flask cultivations. The transformantswere inoculated from the PD slants to shake flasks containing 50 ml ofcomplex lactose-based cellulase inducing medium (Joutsjoki et al., 1993)buffered with 5% KH₂PO₄. The CBHII/Cel6A protein production of thetransformants was analyzed from the culture supernatants after growingthem for 7 days at 30° C., 250 rpm. Heterologous production ofrecombinant proteins was analyzed by SDS-PAGE with subsequent Coomassiestaining. The genotypes of the chosen transformants were confirmed byusing Southern blot analyses in which several genomic digests wereincluded and the respective expression cassette was used as a probe.

The best-producing transformants were chosen to be cultivated inlaboratory scale bioreactors. The transformants were cultivated in labbioreactors at 28° C. in the cellulase inducing complex medium for 3-4days with pH control 4.4±0.2 (NH₃/H₃PO₄) to obtain material for theapplication tests. The supernatants were recovered by centrifugation andfiltering through Seitz-K 150 and EK filters (Pall SeitzSchenkFiltersystems GmbH, Bad Kreuznach, Germany).

Example 3 Hydrolysis of Crystalline Cellulose (Avicel) by theRecombinant CBHII/Cel6A Enzymes

The recombinant CBHII/Cel6A enzyme preparations were characterized interms of pH dependence and thermal stability using crystalline cellulose(Avicel) as a substrate. The pH dependence and thermal stability forrecombinant CBHII/Cel6A proteins from Acremonium thermophilum ALKO4245,Melanocarpus albomyces ALKO4237, Chaetomium thermophilum ALKO4265 andTalaromyces emersonii RF8069 were determined within a pH range of3.0-10.0 and temperature range of 40° C.-80° C., respectively. Thecrystalline cellulose (Ph 101, Avicel; Fluka, Bucsh, Switzerland)hydrolysis assays were performed in 2.0 ml tube scale in 50 mM sodiumacetate pH 6.0. For determination of the pH optima, substrate solutions(Avicel, 50 mg/ml in sodium acetate, pH 6.0) of pH range from 3 to 10were shaken with the enzyme preparations (100 μg protein in reaction) at50° C., and the final volume of the reaction mixture was 650 Thehydrolysis was continued for 21 hours and stopped by adding 326 μl ofstop reagent containing 9 vol of 94% ethanol and 1 vol of 1 M glycine(pH 11). The solution was filtered through a Millex GV13 0.22 μmfiltration unit (Millipore, Billerica, Mass., USA). The formation ofsoluble reducing sugars in the supernatant was determined bypara-hydroxybenzoic-acidhydrazide (PAHBAH) method (Lever, 1972) using acellobiose standard curve (200 μM to 1200 μM cellobiose). A freshly made0.1 M PAHBAH (Sigma-Aldrich, St. Louis, Mo., USA) in 0.5 M NaOH solution(200 μl) was added to 300 μl of the filtered sample and boiled for 10minutes after which the solution was cooled to room temperature. Theabsorbance at 405 nm was measured from duplicate samples by Multiskan EX(Thermo Labsystems, Franklin, Mass., USA). Correspondingly, thermalstability of the recombinant CBHII/Cel6A proteins was determined inAvicel substrate solutions of temperature range from 40° C. to 80° C. atthe optimum pH with the reaction time of 21 hours. Results indicate thatthe pH optimum for Talaromyces emersonii RF8069 CBHII/Cel6A andMelanocarpus albomyces ALKO4237 CBHII/Cel6A is at 4.0, whereasChaetomium thermophilum ALKO4265 and Acremonium thermophilum ALKO4245enzymes have pH optimum at 5.0 (FIG. 2). Thermal stability was found tobe substantially higher for Acremonium thermophilum ALKO4245, Chaetomiumthermophilum ALKO4265 and Talaromyces emersonii RF8069 CBHII/Cel6A ascompared to that of Melanocarpus albomyces ALKO4237 protein (FIG. 3).

Example 4 Hydrolysis of Hardwood Substrate with Enzyme PreparationsComprising a Recombinant CBHII/Cel6A Cellobiohydrolase

Steam exploded hardwood was suspended in 0.05 M sodium acetate buffer,pH 4.8. The final weight of the hydrolysis mixture was 20 g of which thetotal solids concentration was 2% (w/w). The substrate was hydrolysedusing different enzyme mixtures at a dosage of 5 mg of protein per g oftotal solids in 50 ml shake flasks. The protein contents of the enzymecomponents and the mixes were determined using the Pierce BCA assay kit(Thermo Scientific, Product number 23227) with Bovine Serum Albumin(Thermo Scientific, Product number 23209) as standard. The shake flaskswere agitated in a linear-shaking waterbath adjusted in differenttemperatures. For each sample point, a sample of 1 ml was taken fromduplicate shake flasks, and boiled for 10 minutes to terminate theenzymatic hydrolysis, centrifuged, and the supernatant was analysed forreaction products from the hydrolysis. The blanks containing substratealone (only buffer added instead of enzymes) were prepared identicallyto the other samples.

Three separate mixture combinations were prepared (a thermophilicMIXTURE 2, a mesophilic MIXTURE ACC and a mesophilic MIXTURE T. REESEIENZYMES) with different Cel6A/CBHII replacements.

A mixture of thermostable cellulases was prepared using the followingcomponents:

-   -   Thermophilic Cel7A/CBHI preparation containing Thermoascus        aurantiacus ALKO4242 Cel7A with genetically attached CBD of T.        reesei CBH1/Cel7A (WO2007071818).    -   Thermophilic endoglucanase preparation containing Acremonium        thermophilum ALKO4245 Cel45A endoglucanase (At EG_(—)40/Cel45A,        WO2007071818).    -   Thermophilic β-glucosidase preparation containing Thermoascus        aurantiacus ALKO4242 β-glucosidase (Ta βG_(—)81/Cel3A,        WO2007071818).    -   Thermophilic xylanase preparation containing Nonomurea flexuosa        Xyn11A (AM24, WO2005100557, AB Enzymes Oy, FI)).

All cellulases were heterologously produced as monocomponents inTrichoderma reesei host strain having cellulase-free background (thegenes encoding the four major cellulases Cel7A/CBHI, Cel6A/CBHII,Cel7B/EGI and Cel5A/EGII were deleted). Crude culture supernatants wereused in the mixture. The enzyme components were combined as follows (per10 ml of mixture): CBHI/Cel7A preparation 330 mg (71.2%), endoglucanasepreparation 105 mg (22.7%), β-glucosidase preparation 7.5 mg (1.6%) andxylanase preparation 21 mg (4.5%). The volume was made up to 10 ml withtap water. The final protein concentration of the mixture was 46.35mg/ml. This enzyme mixture was designated as MIXTURE 2.

For testing Cel6A/CBHII performance in the hydrolysis with MIXTURE 2,15% (49.5 mg) of the CBHI/Cel7A component of MIXTURE 2 was replaced byMa_ALKO4237_Cel6A (MIXTURE 2_MA), Ct_ALKO4265_Cel6A (MIXTURE 2_CT), orAt_ALKO4245_Cel6A (MIXTURE 2_AT), respectively.

A state-of-the-art mixture was prepared by combining the followingcomponents (per 10 ml of mixture): ECONASE® CE (Roal Oy, a classical T.reesei enzyme product) 470 mg (94%), β-glucosidase preparation (AtβG_(—)101/Cel3A, WO2007071818) 20 mg (4%) and xylanase preparation (TaXYN_(—)30, WO2007071818) 10 mg (2%). The volume was made up to 10 mlwith tap water. The final protein concentration in this mixture was 50mg/ml. This enzyme mixture was designated as MIXTURE T. REESEI ENZYMES.At βG_(—)101/Cel3A and Ta XYN_(—)30 enzymes were heterologously producedas monocomponents in Trichoderma reesei host strain havingcellulase-free background.

For testing Cel6A/CBHII performance in the hydrolysis with MIXTURE T.REESEI ENZYMES, 15% (70.5 mg) of the ECONASE® CE component of MIXTURE T.REESEI ENZYMES was replaced by Ma_ALKO4237Cel6A (MIXTURE TR_MA),Ct_ALKO4265_Cel6A (MIXTURE TR_CT), or At_ALKO4245_Cel6A (MIXTURE TR_AT),respectively.

MIXTURE ACC was prepared from commercial Accellerase® 1000 (fromGenencor International/Danisco A/S) product (per 10 ml): Accellerase®1000 400 mg protein (100%). The volume was made up to 10 ml with tapwater. The final protein concentration in this mixture was 40 mg/ml.

For testing Cel6A/CBHII performance in the hydrolysis with MIXTURE ACC,15% (60 mg) of the Accellerase® 1000 component of MIXTURE ACC wasreplaced by Ma_ALKO4237_Cel6A (MIXTURE ACC_MA), Ct_ALKO4265_Cel6A(MIXTURE ACC_CT), or At_ALKO4245_Cel6A (MIXTURE ACC_AT), respectively.

For MIXTURE 2 combinations, the hydrolysis was performed at 55° C.,while the hydrolysis temperature for MIXTURE T. REESEI ENZYMES andMIXTURE ACC experiments was 37° C. Samples were taken from thehydrolysis after 72 h, quantified by HPLC and the concentrations ofglucose and xylose were determined. The results from the substrateblanks were subtracted from the samples with enzymes, and theconcentration of glucose and xylose combined is shown in FIG. 4A-C.

The results clearly show better performance of the MIXTURE 2 with thethermostable Cel6A/CBHII enzymes at 55° C. The amount of sugars releasedfrom the hardwood substrate was found to increase 12%, 14% and 26% bysupplementing with Ma_ALKO4237_Cel6A, Ct_ALKO4265_Cel6A orAt_ALKO4245_Cel6A enzymes in the MIXTURE 2, respectively. Acremoniumthermophilum ALKO4245 enzyme was found to be best-performing Cel6A/CBHIIherein studied (FIG. 4A). At_ALKO4245_Cel6A shows increased hydrolysisalso at 37° C. added either in the state-of-the-art Trichoderma mixture(MIXTURE T. REESEI ENZYMES) (FIG. 4B) or in the commercial product(MIXTURE ACC) (FIG. 4C).

Example 5 Hydrolysis of Corn Cobs with Enzyme Preparations Comprising aRecombinant CBHII/Cel6A Cellobiohydrolase

Steam exploded corn cobbs was suspended in 0.05 M sodium acetate buffer,pH 4.8. The final weight of the hydrolysis mixture was 20 g of which thetotal solids concentration was 2% (w/w). The substrate was hydrolysedusing different enzyme mixtures at a dosage of 5 mg of protein per g oftotal solids in 50 ml shake flasks. The protein contents of the enzymecomponents and the mixes were determined using the Pierce BCA assay kit(Thermo Scientific, Product number 23227) with Bovine Serum Albumin(Thermo Scientific, Product number 23209) as standard. The shake flaskswere agitated in a linear-shaking waterbath adjusted at 55° C. For eachsample point, a sample of 1 ml was taken from duplicate shake flasks,and boiled for 10 minutes to terminate the enzymatic hydrolysis,centrifuged, and the supernatant was analysed for reaction products fromthe hydrolysis. The blanks containing substrate alone (only buffer addedinstead of enzymes) were prepared identically to the other samples.

A mixture of thermostable cellulases was prepared using the followingcomponents:

-   -   Thermophilic Cel7A/CBHI preparation containing Thermoascus        aurantiacus ALKO4242 Cel7A with genetically attached CBD of T.        reesei CBHI/Cel7A (WO2007071818).    -   Thermophilic endoglucanase preparation containing Acremonium        thermophilum ALKO4245 Cel45A endoglucanase (At EG_(—)40/Cel45A,        WO2007071818).    -   Thermophilic β-glucosidase preparation containing Thermoascus        aurantiacus ALKO4242 β-glucosidase (Ta βG_(—)81/Cel3A,        WO2007071818).    -   Thermophilic xylanase preparation containing Nonomurea flexuosa        Xyn11A (AM24, WO2005100557).

All cellulases were heterologously produced as monocomponents inTrichoderma reesei host strain having cellulase-free background (thegenes encoding the four major cellulases Cel7A/CBHI, Cel6A/CBHII,Cel7B/EGI and Cel5A/EGII were deleted). Crude culture supernatants wereused in the mixture. The enzyme components were combined as follows (per10 ml of mixture): CBHI/Cel7A preparation 330 mg (71.2%), endoglucanasepreparation 105 mg (22.7%), β-glucosidase preparation 7.5 mg (1.6%) andxylanase preparation 21 mg (4.5%). The volume was made up to 10 ml withtap water. The final protein concentration of the mixture was 46.35mg/ml. This enzyme mixture was designated as MIXTURE 2.

For testing At_ALKO4245_Cel6A performance in the hydrolysis with MIXTURE2, 15% (49.5 mg) of the CBHI/Cel7A component of MIXTURE 2 was replacedby At_ALKO4245_Cel6A (MIXTURE 2_AT), respectively.

Samples were taken from the hydrolysis after 72 h, quantified by HPLCand the concentrations of glucose and xylose were determined. Theresults from the substrate blanks were subtracted from the samples withenzymes, and the concentration of glucose and xylose combined is shownin FIG. 5.

Similar to that described in the Example 4, the results here clearlyshow better performance of the MIXTURE 2 with the At_ALKO4245_Cel6Aenzyme at 55° C. for the corn cobs substrate.

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1. A method for treating cellulosic material with a CBHII/Cel6A polypeptide or an enzyme preparation comprising said polypeptide, wherein said CBHII/Cel6A polypeptide has cellobiohydrolase activity and comprises an amino acid sequence having at least 76% identity to the full-length polypeptide of SEQ ID NO: 12, at least 76% identity to the full-length polypeptide of SEQ ID NO:14, at least 95% identity to the full-length polypeptide of SEQ ID NO:15, or at least 91% identity to the full-length polypeptide of SEQ ID NO:16, or a fragment or variant thereof having similar properties, and wherein said method comprises the following steps: i) producing said CBHII/Cel6A polypeptide or an enzyme preparation comprising said polypeptide or a fermentative microorganism producing said polypeptide; ii) reacting the cellulosic material with said CBHII/Cel6A polypeptide or the enzyme preparation comprising said polypeptide or the fermentative microorganism producing said polypeptide; and iii) obtaining partially or fully hydrolyzed cellulosic material.
 2. The method according to claim 1, wherein the CBHII/Cel6A polypeptide is obtainable from a genus of Acremonium, Melanocarpus, Chaetomium or Talaromyces, more preferably from A. thermophilum, M albomyces, C. thermophilum or T. emersonii, most preferably from the deposited strain A. thermophilum CBS 116240, M. albomyces CBS 685.95, C. thermophilum CBS 730.95 or T. emersonii DSM
 2432. 3. The method according to claim 1, wherein the CBHII/Cel6A polypeptide has the amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO:
 16. 4. The method according to claim 1, wherein the CBHII/Cel6A polypeptide is capable of hydrolyzing cellulosic material at moderate to elevated temperatures.
 5. The method according to claim 1, wherein the cellulosic material is treated with an enzyme composition comprising one or more of said CBHII/Cel6A polypeptides in combination with at least one further enzyme capable of hydrolyzing said cellulosic material selected from a group of cellobiohydrolase, endoglucanase, beta-glucosidase, beta-glucanase, xyloglucanase, xylanase, beta-xylosidase, mannanase, beta-mannosidase, α-glucuronidase, acetyl xylan esterase, α-arabinofuranosidase, α-galactosidase, pectinase, involving endo- and exo-α-L-arabinases, α-galactosidase, endo- and exo-galactoronase, endopectinlyase, pectate lyase, and pectinesterase, phenol esterase, ligninase involving lignin peroxidase, manganese-dependent peroxidase, H₂O₂-generating enzyme and laccase with or without mediators.
 6. The method according to claim 5, wherein the enzymes are added to the cellulosic material either simultaneously or sequentially.
 7. The method according to claim 1, wherein the cellulosic material is selected from the group consisting of plant materials, agricultural biomass, waste products and dedicated energy crops.
 8. The method according to claim 1, wherein said CBHII/Cel6A polypeptide is applicable in production of biofuel.
 9. The method according to claim 1, wherein said CBHII/Cel6A polypeptide derives from A. thermophilum CBS 116240 and has the amino acid sequence of SEQ ID NO:12.
 10. A fungal CBHII/Cel6A polypeptide, wherein said polypeptide has cellobiohydrolase activity and comprises an amino acid sequence having at least 76% identity to the full-length polypeptide of SEQ ID NO: 12, at least 76% identity to SEQ ID NO: 14, at least 95% identity to SEQ ID NO: 15, or at least 91% identity to SEQ ID NO: 16, or a fragment or variant thereof having similar properties.
 11. The CBHII/Cel6A polypeptide according to claim 10, wherein said polypeptide is obtainable from a genus of Acremonium, Melanocarpus, Chaetomium or Talaromyces, more preferably from species A. thermophilum, M albomyces, C. thermophilum or T. emersonii, most preferably from A. thermophilum CBS 116240, M. albomyces CBS 685.95, C. thermophilum CBS 730.95 or T. emersonii DSM
 2432. 12. The CBHII/Cel6A polypeptide according to claim 10, wherein said polypeptide has the amino acid sequence of SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15 or SEQ ID NO:16.
 13. The CBHII/Cel6A polypeptide according to claim 10, wherein said polypeptide is capable of hydrolyzing cellulosic material at moderate to elevated temperatures.
 14. The CBHII/Cel6A polypeptide according to claim 10, wherein said polypeptide is encoded by an isolated nucleic acid molecule, which comprises a polynucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15 or SEQ ID NO:16 or an amino acid sequence having at least 76% identity to the amino acid sequence of the full-length polypeptide SEQ ID NO:12, at least 76% identity to SEQ ID NO:14, at least 95% identity to SEQ ID NO:15 or at least 91% identity to SEQ ID NO:16, or a fragment or variant thereof having similar properties.
 15. The CBHII/Cel6A polypeptide according to claim 10, wherein said polypeptide is encoded by an isolated nucleic acid molecule comprising a polynucleotide sequence included in SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:9 or SEQ ID NO:10 or a subsequence thereof.
 16. The CBHII/Cel6A polypeptide according to claim 10, wherein said polypeptide is encoded by an isolated nucleic acid molecule, which hybridizes under stringent conditions to a polynucleotide sequence included in SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:7 or SEQ ID NO:8 or a subsequence thereof.
 17. The CBHII/Cel6A polypeptide according to claim 10, wherein said polypeptide is encoded by an isolated nucleic acid molecule comprising a polynucleotide sequence included in SEQ ID NO:
 11. 18. The CBHII/Cel6A polypeptide according to claim 10, wherein said polypeptide is encoded by the polynucleotide sequence included in plasmid pALK2582 deposited in Escherichia coli under accession number DSM 22946, plasmid pALK2581 deposited in Escherichia coli under accession number DSM 22945, plasmid pALK2904 deposited in Escherichia coli under accession number DSM 22947 or plasmid pALK3006 deposited in Escherichia coli under accession number DSM
 23185. 19. The CBHII/Cel6A polypeptide according to claim 10, wherein said polypeptide is produced from a recombinant expression vector comprising a nucleic acid molecule comprising a polynucleotide sequence encoding a CBHII/Cel6A enzyme of claim 10, and being operably linked to regulatory sequences capable of directing expression of the CBHII/Cel6A encoding nucleic acid molecule in a suitable host.
 20. The CBHII/Cel6A polypeptide according to claim 10, wherein said polypeptide is produced in a heterologous host, preferably in a microbial host.
 21. The CBHII/Cel6A polypeptide according to claim 10, wherein said polypeptide is produced in a host of genus Trichoderma, Aspergillus, Fusarium, Humicola, Chrysosporium, Neurospora, Rhizopus, Penicillium or Mortiriella.
 22. The CBHII/Cel6A polypeptide according to claim 21, wherein said polypeptide is produced in Trichoderma or Aspergillus, preferably in Trichoderma reesei.
 23. The CBHII/Cel6A polypeptide according to claim 10, wherein said CBHII/Cel6A polypeptide derives from A. thermophilum CBS 116240 and has the amino acid sequence of SEQ ID NO:
 12. 24. An isolated nucleic acid molecule, which comprises a polynucleotide sequence encoding a fungal CBHII/Cel6A polypeptide selected from the group consisting of: (a) a nucleic acid molecule, which comprises a polynucleotide sequence encoding a polypeptide having cellobiohydrolase activity and comprising the full-length amino acid sequence as depicted in SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15 or SEQ ID NO:16, or a fragment or variant thereof having similar properties; (b) a nucleic acid molecule, which comprises a polynucleotide sequence encoding a polypeptide having cellobiohydrolase activity and at least 76% identity to the full-length amino acid sequence of SEQ ID NO:12, at least 76% identity to the full-length amino acid sequence of SEQ ID NO:14, at least 95% identity to the full-length amino acid sequence of SEQ ID NO:15 or at least 91% identity to the full-length amino acid sequence of SEQ ID NO:16, or a fragment or variant thereof having similar properties; c) a nucleic acid molecule comprising the coding sequence of the polynucleotide sequence depicted as SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:9 or SEQ ID NO:10; (d) a nucleic acid molecule comprising the coding sequence of the polynucleotide sequence contained in DSM 22946, DSM 22945, DSM 22947 or DSM 23185; (e) a nucleic acid molecule the coding sequence of which differs from the coding sequence of a nucleic acid molecule of any one of (c) to (d) due to the degeneracy of the genetic code; and (f) a nucleic acid molecule hybridizing under stringent conditions to a nucleic acid molecule contained in DSM 22946, DSM 22945, DSM 22947 or DSM 23185, and encoding a polypeptide having cellobiohydrolase activity and an amino acid sequence which shows at least 76% identity to the full-length amino acid sequence as depicted in SEQ ID NO:12, at least 76% identity to the full-length amino acid sequence of SEQ ID NO:14, at least 95% identity to the full-length amino acid sequence of SEQ ID NO:15 or at least 91% identity to the full-length amino acid sequence of SEQ ID NO:16, or a fragment or variant thereof having similar properties.
 25. The nucleic acid molecule according to claim 24, wherein said CBHII/Cel6A encoding nucleic acid molecule derives from A. thermophilum CBS 116240 and has the polynucleotide sequence of SEQ ID NO:11.
 26. A recombinant expression vector comprising the nucleic acid molecule of claim 24 operably linked to regulatory sequences capable of directing expression of the CBHII/Cel6A cellobiohydrolase gene and production of said CBHII/Cel6A cellobiohydrolase in a suitable host.
 27. A host cell comprising the recombinant expression vector of claim
 26. 28. The host cell according to claim 27, wherein said host is a microbial host.
 29. The host cell according to claim 28, wherein said host is a filamentous fungus.
 30. The host cell according to claim 29, wherein said host is of a genus Trichoderma, Aspergillus, Fusarium, Humicola, Chrysosporium, Neurospora, Rhizopus, Penicillium and Mortiriella.
 31. The host cell according to claim 30, wherein said host is Trichoderma or Aspergillus, preferably Trichoderma reesei.
 32. The host cell according claim 27, wherein said host is a fermentative microorganism.
 33. A process of producing a polypeptide having cellobiohydrolase activity, said process comprising the steps of culturing the host cell of claim 27 and recovering the polypeptide.
 34. A polypeptide having cellobiohydrolase activity encoded by the nucleic acid sequence of claim 24 and which is obtainable by the process of claim
 33. 35. A process for obtaining an enzyme preparation, said process comprising the steps of culturing a host cell of claim 27 and preparing a whole culture broth, or separating the cells from a spent culture medium and obtaining supernatant.
 36. An enzyme preparation obtainable by the process according to claim
 35. 37. An enzyme preparation, which comprises the CBHII/Cel6A enzyme according to claim
 10. 38. The enzyme preparation according to claim 37, wherein the CBHII/Cel6A cellobiohydrolase derives from Acremonium thermophilum and has the amino acid sequence of SEQ ID NO:
 12. 39. The enzyme preparation according to claim 36, wherein said preparation comprises other enzymes selected from the group of cellobiohydrolase, endoglucanase, beta-glucosidase, beta-glucanase, xyloglucanase, xylanase, beta-xylosidase, mannanase, beta-mannosidase, α-glucuronidase, acetyl xylan esterase, α-arabinofuranosidase, α-galactosidase, pectinase, involving endo- and exo-α-L-arabinases, α-galactosidase, endo- and exo-galactoronase, endopectinlyase, pectate lyase and pectinesterase, phenol esterase, ligninase involving lignin peroxidase, manganese-dependent peroxidase, H₂O₂-generating enzyme and laccase with or without a mediator.
 40. The enzyme preparation according to claim 36, wherein said enzyme preparation is in a form of whole culture broth, spent culture medium, liquid, powder or granulate.
 41. Use of the CBHII/Cel6A polypeptide of claim 10 or the enzyme preparation of claim 36 for production of biofuel, for detergents, for treating fibers, for treating food or feed, for pulp and paper, for beverage or for any applications involving hydrolysis or modification of cellulosic material.
 42. Use of the CBHII/Cel6A polypeptide of claim 10 or the enzyme preparation of claim 36 for production of biofuel. 